U.S. patent application number 10/588687 was filed with the patent office on 2008-01-31 for therapeutic molecules and methods for generating and/or selecting same.
Invention is credited to Jerry M. Adams, Lin Chen, Peter M. Colman, Catherine L. Day, Mark G. Hinds, David C. S. Huang, Brian J. Smith, Andrew Wei, Simon N. Willis.
Application Number | 20080027145 10/588687 |
Document ID | / |
Family ID | 34831683 |
Filed Date | 2008-01-31 |
United States Patent
Application |
20080027145 |
Kind Code |
A1 |
Huang; David C. S. ; et
al. |
January 31, 2008 |
Therapeutic Molecules and Methods for Generating and/or Selecting
Same
Abstract
The present invention relates generally to therapeutic molecules
useful for inducing apoptosis of particular cells such as, but not
limited to, cancer cells and methods for generating and/or
selecting same. The present invention further provides methods for
inducing apoptosis of cells such as cancer cells and pharmaceutical
compositions useful for same. The present invention further
provides methods for generating or selecting therapeutic agents
capable of inducing apoptosis of particular cells by the selective
inhibition of pro-survival proteins. The present invention further
provides a computational approach to therapeutic molecule design
based on structure-binding characteristics.
Inventors: |
Huang; David C. S.; (North
Fitzroy, AU) ; Colman; Peter M.; (East Melbourne,
AU) ; Day; Catherine L.; (Maori Hill, NZ) ;
Adams; Jerry M.; (North Melbourne, AU) ; Chen;
Lin; (Rosanna East, AU) ; Willis; Simon N.;
(Keilor, AU) ; Wei; Andrew; (Blackburn, AU)
; Smith; Brian J.; (Sunbury, AU) ; Hinds; Mark
G.; (Brunswick West, AU) |
Correspondence
Address: |
THE MCCALLUM LAW FIRM, P. C.
685 BRIGGS STREET, PO BOX 929
ERIE
CO
80516
US
|
Family ID: |
34831683 |
Appl. No.: |
10/588687 |
Filed: |
February 3, 2005 |
PCT Filed: |
February 3, 2005 |
PCT NO: |
PCT/AU05/00140 |
371 Date: |
July 18, 2007 |
Current U.S.
Class: |
514/789 ;
436/501; 702/19 |
Current CPC
Class: |
C07K 14/4747 20130101;
A61P 35/00 20180101 |
Class at
Publication: |
514/789 ;
436/501; 702/19 |
International
Class: |
A61K 35/00 20060101
A61K035/00; A61P 35/00 20060101 A61P035/00; G01N 33/48 20060101
G01N033/48; G01N 33/53 20060101 G01N033/53 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 6, 2004 |
AU |
2004900562 |
Claims
1-18. (canceled)
19. A method for generating an antagonist of the pro-survival Bcl-2
family, said method comprising; selecting a scaffold BH3-only
protein structure with residue positions defining an amphipathic
a-helix formed by the BH3 domain; selecting one or more residue
positions associated with a promiscuous binding phenotype of a
BH3-only protein; substituting amino acid residues conferring a
promiscuous phenotype for a molecule selected from the group
consisting of amino acids and amino acid chemical analogs which
confer a restrictive binding pattern to a Bcl-2 protein; and
analyzing the interaction of each substitution for an ability to
induce a more restrictive spectrum of binding to a Bcl-2
protein.
20. The method of claim 19 wherein said Bcl-2 antagonist is
specific for one or more molecules selected from the group
consisting of Bcl-2, Bcl-x.sub.L, Bcl-w, Mcl and A1.
21. The method of claim 19 wherein said Bcl-2 antagonist is based
on the structure of a molecule selected from the group consisting
of Noxa, Bim, Puma, Bmf, Bad, Bik, Hrk and Bid.
22. The method of claim 19 wherein said Bcl-2 antagonist inhibits a
pro-survival protein on a cancer cell.
23. The method of claim 22 wherein said cancer cell is selected
from the group consisting of ABL 1 protooncogene, AIDS Related
Cancers, Acoustic Neuroma, Acute Lymphocytic Leukaemia, Acute
Myeloid Leukaemia, Adenocystic carcinoma, Adrenocortical Cancer,
Agnogenic myeloid metaplasia, Alopecia, Alveolar soft-part sarcoma,
Anal cancer, Angiosarcoma, Aplastic Anaemia, Astrocytoma,
Ataxia-telangiectasia, Basal Cell Carcinoma (Skin), Bladder Cancer,
Bone Cancers, Bowel cancer, Brain Stem Glioma, Brain and CNS
Tumors, Breast Cancer, CNS Tumors, Carcinoid Tumors, Cervical
Cancer, Childhood Brain Tumors, Childhood Cancer, Childhood
Leukaemia, Childhood Soft Tissue Sarcoma, Chondrosarcoma,
Choriocarcinoma, Chronic Lymphocytic Leukaemia, Chronic Myeloid
Leukaemia, Colorectal Cancers, Cutaneous T-Cell Lymphoma,
Dermatofibrosarcoma-protuberans,
Desmoplastic-Small-Round-Cell-Tumor, Ductal Carcinoma, Endocrine
Cancers, Endometrial Cancer, Ependymoma, Esophageal Cancer, Ewing's
Sarcoma, Extra-Hepatic Bile Duct Cancer, Eye Cancer, Eye: Melanoma,
Retinoblastoma, Fallopian Tube cancer, Fanconi Anaemia,
Fibrosarcoma, Gall Bladder Cancer, Gastric Cancer, Gastrointestinal
Cancers, Gastrointestinal-Carcinoid-Tumor, Genitourinary Cancers,
Germ Cell Tumors, Gestational-Trophoblastic-Disease, Glioma,
Gynaecological Cancers, Haematological Malignancies, Hairy Cell
Leukaemia, Head and Neck Cancer, Hepatocellular Cancer, Hereditary
Breast Cancer, Histiocytosis, Hodgkin's Disease, Human
Papillomavirus, Hydatidiform mole, Hypercalcemia, Hypopharynx
Cancer, IntraOcular Melanoma, Islet cell cancer, Kapos's sarcoma,
Kidney Cancer, Langerhan's-Cell-Histiocytosis, Laryngeal Cancer,
Leiomyosarcoma, Leukaemia, Li-Fraumeni Syndrome, Lip Cancer,
Liposarcoma, Liver Cancer, Lung Cancer, Lymphedema, Lymphoma,
Hodgkin's Lymphoma, Non-Hodgkin's Lymphoma, Male Breast Cancer,
Malignant-Rhabdoid-Tumor-of-Kidney, Medulloblastoma, Melanoma,
Merkel Cell Cancer, Mesothelioma, Metastatic Cancer, Mouth Cancer,
Multiple Endocrine Neoplasia, Mycosis Fungoides, Myelodysplastic
Syndromes, Myeloma, Myeloproliferative Disorders, Nasal Cancer,
Nasopharyngeal Cancer, Nephroblastoma, Neuroblastoma,
Neurofibromatosis, Nijmegen Breakage Syndrome, Non-Melanoma Skin
Cancer, Non-Small-Cell-Lung-Cancer-(NSCLC), Ocular Cancers,
Oesophageal Cancer, Oral cavity Cancer, Oropharynx Cancer,
Osteosarcoma, Ostomy Ovarian Cancer, Pancreas Cancer, Paranasal
Cancer, Parathyroid Cancer, Parotid Gland Cancer, Penile Cancer,
Peripheral-Neuroectodermal-Tumors, Pituitary Cancer, Polycythemia
vera, Prostate Cancer, Rare-cancers-and-associated-disorders, Renal
Cell Carcinoma, Retinoblastoma, Rhabdomyosarcoma, Rothmund-Thomson
Syndrome, Salivary Gland Cancer, Sarcoma, Schwannoma, Sezary
syndrome, Skin Cancer, Small Cell Lung Cancer (SCLC), Small
Intestine Cancer, Soft Tissue Sarcoma, Spinal Cord Tumors,
Squamous-Cell-Carcinoma-(skin), Stomach Cancer, Synovial sarcoma,
Testicular Cancer, Thymus Cancer, Thyroid Cancer,
Transitional-Cell-Cancer-(bladder),
Transitional-Cell-Cancer-(renal-pelvis-/-ureter), Trophoblastic
Cancer, Urethral Cancer, Urinary System Cancer, Uroplakins, Uterine
sarcoma, Uterus Cancer, Vaginal Cancer, Vulva Cancer,
Waldenstrom's-Macroglobulinemia, and Wilms'Tumor.
24. The method of claim 19 wherein said Bcl-2 antagonist inhibits a
prosurvival protein in a mammal.
25. The method of claim 24, wherein said mammal is a human.
26. A method for generating an antagonist of the pro-survival Bcl-2
protein family, said method comprising; selecting a restrictive
BH3-only protein as a scaffold protein; determining the
conformation of the scaffold conferring the restrictive phenotype;
and generating a chemical compound which mimics said scaffold and
conformational part conferring a restrictive spectrum of binding to
a Bcl-2 protein.
27. A method for selecting an antagonist of the pro-survival Bcl-2
protein family, said method comprising; selecting a restrictive
BH3-only protein as a scaffold protein; determining the
conformation of the scaffold conferring the restrictive phenotype;
and generating a chemical compound which mimics said scaffold and
conformational part conferring a restrictive spectrum of binding to
a Bcl-2 protein.
28. A computational method for designing an antagonist of the
pro-survival Bcl-2 protein family based on a scaffold BH3-only
protein with residue positions conferring a restrictive phenotype
comprising; selecting a collection of promiscuous BH3- only
proteins; providing a sequence alignment of said proteins and
comparing said sequence alignment to a restrictive BH3-only
protein; generating a frequency of occurrence for individual amino
acids in one or a plurality of positions with said alignments
conferring promiscuity or restrictivity with respect to binding to
Bcl-2 proteins; creating a scoring function selected from the group
consisting of charge, size, conformation, solubility, polarity,
hydrophobicity, hydrophilicity and contribution to tertiary
structure using said frequencies; using said scoring function and
at least one additional scoring function to generate information
selected from the group consisting of a set of optimized protein
sequences and the conformational equivalents of optimized protein
sequences; and performing an activity selected from the group
consisting of generating a compound having a restrictive binding
phenotype to a Bcl-2 protein and selecting a protein having a
restrictive binding phenotype to a Bcl-2 protein.
29. The method of claim 28 wherein said scoring function is
selected from the group consisting of the number and position of
acidic residues, the number and position of basic residues, the
number and position of polar residues, the number and position of
non-polar residues, the number and position of charged residues,
the number and position of uncharged residues, the number and
position of hydrophilic residues, the number and position of
hydrophobic residues, the levels of residues, the solubility levels
of residues, the size of residues, and the contribution to tertiary
structure the residue makes in the BH3-only protein.
30. A computer program product for determining the structure of an
agent to induce apoptosis in a cell, said product comprising: (1)
code that receives as input scoring function (SF) for at least two
features associated with a molecule selected from the group
consisting of BH3-only and Bcl-2, wherein said features are
selected from the group consisting of: (m) the number and position
of acidic residues, (n) the number and position of basic residues,
(o) the number and position of polar residues, (p) the number and
position of non-polar residues, (q) the number and position of
charged residues, (r) the number and position of uncharged
residues, (s) the number and position of hydrophillic residues, (t)
the number and position of hydrophobic residues, (u) the levels of
residues, (v) the solubility levels of residues, (w) the size of
residues, and (x) the contribution to tertiary structure the
residue makes in the BH3-only protein; (2) code that adds said SF
to provide a sum corresponding to a Pv for BH3-only proteins; and
(3) a computer readable medium that stores the codes.
31. An apparatus for assessing the likely usefulness of a BH3-only
protein or chemical equivalent to induce apoptosis in a cell
comprising; (1) a machine-readable data storage medium comprising a
data storage material encoded with machine-readable data, wherein
said machine-readable data comprise lys for at least two features
associated with a molecule selected from the group consisting of
BH3-only and Bcl-2, wherein said features are selected from the
group consisting of: (m) the number and position of acidic
residues, (n) the number and position of basic residues, (o) the
number and position of polar residues, (p) the number and position
of non-polar residues, (q) the number and position of charged
residues, (r) the number and position of uncharged residues, (s)
the number and position of hydrophillic residues, (t) the number
and position of hydrophobic residues, (u) the levels of residues,
(v) the solubility levels of residues, (w) the size of residues,
and (x) the contribution to tertiary structure the residue makes in
the BH3-only protein; (2) a working memory for storing instructions
for processing said machine-readable data; (3) a central-processing
unit coupled to said working memory and to said machine-readable
data storage medium, for processing said machine readable data to
provide a sum of said SF corresponding to a Pv for said compound
(s); and (4) an output hardware coupled to said central processing
unit, for receiving said Pv.
32. A method of treating cancer in a subject comprising;
administering to said subject an effective amount of an antagonist
of a Bcl-2 protein for a time and under conditions sufficient to
decrease said cancer.
33. The method of claim 32, further comprising one or more
pharmaceutically acceptable carriers or diluents.
34. The method of claim 32 wherein administration of said
antagonist is accomplished by a method selected from the group
consisting of respiratoral, intratracheal, nasopharyngeal,
intravenous, intraperitoneal, subcutaneous, intracranial,
intradermal, intramuscular, intraoccular, intrathecal,
intracereberal, intranasal, infusion, oral, rectal, patch and
implant rates.
35. A method of preventing cancer in a subject comprising;
administering to said subject an effective amount of an antagonist
of a Bcl-2 protein for a time and under conditions sufficient to
prevent said cancer.
36. The method of claim 35 further comprising one or more
pharmaceutically acceptable carriers or diluents.
37. The method of claim 35 wherein administration of said
antagonist is accomplished by a method selected from the group
consisting of respiratoral, intratracheal, nasopharyngeal,
intravenous, intraperitoneal, subcutaneous, intracranial,
intradermal, intramuscular, intraoccular, intrathecal,
intracereberal, intranasal, infusion, oral, rectal, patch and
implant rates.
38. A composition comprising; (a) an antagonist of the pro-survival
Bcl-2 protein family said antagonist generated by the method of a
scaffold BH3-only protein structure with residue positions defining
an amphipathic .alpha.-helix formed by the BH3 domain; (b)
selecting one or more residue positions associated with a
promiscuous binding phenotype of a BH3-only protein; (c)
substituting amino acid residues conferring a promiscuous phenotype
for amino acids or their chemical analogs which confer a
restrictive binding pattern to a Bcl-2 protein; and (d) analyzing
the interaction of each substitution for an ability to induce a
more restrictive spectrum of binding to a Bcl-2 protein, said
composition further comprising one or more pharmaceutically
acceptable carriers and/or diluents.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates generally to therapeutic
molecules useful for inducing apoptosis of particular cells such
as, but not limited to, cancer cells and methods for generating
and/or selecting same. The present invention further provides
methods for inducing apoptosis of cells such as cancer cells and
pharmaceutical compositions useful for same. The present invention
further provides methods for generating or selecting therapeutic
agents capable of inducing apoptosis of particular cells by the
selective inhibition of pro-survival proteins. The present
invention further provides a computational approach to therapeutic
molecule design based on structure-binding characteristics.
[0003] 2. Description of the Prior Art
[0004] Reference to any prior art in this specification is not, and
should not be taken as, an acknowledgment or any form of suggestion
that this prior art forms part of the common general knowledge in
any country.
[0005] Bibliographic details of references provided in this
document are listed at the end of the specification.
[0006] Cancer is the second leading cause of death in the developed
world. Apart from the suffering it causes to patients and their
families it is also one of the most expensive diseases to treat
(Zhang, Nat Rev Drug Discov 1:101-102, 2002). Accordingly,
notwithstanding the toll on human life, if both treatment costs and
the cost of reduced economic productivity are considered, the total
annual economic burden to society is expected to be in the order of
US$200-500 billion by 2010.
[0007] Perturbation of programmed cell death (apoptosis) is a
central step in the development of many major diseases including
cancer. One family of critical regulators of apoptosis is the Bcl-2
protein family. Studies have shown that Bcl-2 overexpression,
enforced in human follicular lymphoma, inhibits apoptosis and
contributes to tumorigenesis (Vaux et al., Nature 335:440-442,
1988; Strasser et al., Nature 348:331-333, 1990). Bcl-2
overexpression has also been noted in up to 90% of breast, colonic
and prostatic cancers (Zhang, 2002, Supra), which represent some of
the most common cancers. Pro-survival relatives of Bcl-2 are also
overexpressed in many tumors. Indeed, impaired apoptosis is now
accepted as a central step in the development of most forms of
malignancy (Cory et al., Nat Rev Cancer 2:647-656, 2002).
[0008] Impaired apoptosis is also a major impediment to the
efficacy of cytotoxic cancer therapy (Cory et al., 2002, Supra;
Johnstone et al., Cell 108:153-164, 2002). Most cytotoxic agents,
including many chemotherapeutic drugs and radiation, indirectly
trigger apoptosis through molecules such as the tumor suppressor
p53 (Cory et al., 2002, Supra). In most tumors, however, the p53
pathway is inactivated, preventing the signals to initiate
apoptosis. Hence, either loss of p53 function or overexpression of
Bcl-2 can provoke chemoresistance, a common cause for treatment
failure.
[0009] Those members of the Bcl-2 protein family that promote cell
survival, including mammalian Bcl-2, Bcl-x.sub.L, Bcl-w, Mcl-1 and
A1, contain three or four BH (B3cl-2 homology) regions of sequence
similarity, and finction until neutralized by their BH3-only
relatives. These pro-apoptotic antagonists, which include mammalian
Bim, Puma, Bmf, Bad, Bik, Hrk, Bid and Noxa, are related to each
other and the wider family only by the short BH3 domain (Huang and
Strasser, Cell 103:839-842, 2000). In contrast, Bax and Bak, a
sub-group of pro-apoptotic family members, share three BH domains
with Bcl-2 and have an essential downstream role, probably in
permeabilization of intracellular membranes (Wei et al., Science
292:727-730, 2001).
[0010] The BH3-only proteins monitor cellular well-being and damage
signals trigger their binding to pro-survival Bcl-2-like proteins,
thereby initiating cell death (Cory et al., Oncogene 22:8590-8607,
2003; Huang and Strasser, 2000, Supra). Their differential
activation, induced by transcriptional cues (e.g. Bim, Puma, Noxa)
or various post-translational mechanisms (e.g. Bim, Bmf, Bad, Bid),
imparts some signaling specificity (Puthalakath et al., Cell Death
Differ 9:505-512, 2002). Once activated, however, the various
BH3-only proteins are generally thought to function similarly by
targeting all the pro-survival Bcl-2-like proteins (Adams et al.,
Genes Dev 17:2481-2495, 2003; Cory et al., Oncogene 22:8590-8607,
2003; Huang and Strasser, 2000, Supra). Their interactions have
not, however, been systematically characterized, and the few
quantitative studies have been confined to Bcl-x.sub.L or Bcl-2
(Letai et al., Cancer Cell 2:183-192, 2002; Petros et al., 2000,
Supra; Sattler et al., 1997, Supra). Establishing whether the
diverse BH3-only proteins and pro-survival family members interact
selectively or promiscuously is important for clarifying how cell
death initiates (Adams, 2003, Supra; Cory et al., 2003, Supra;
Danial and Korsmeyer, Cell 116:205-219, 2004) and is very pertinent
to current efforts to develop compounds that mimic the action of
BH3-only proteins as novel anti-cancer agents.
[0011] In light of the requirement for less toxic and better
targeted anticancer therapies, there is a clear need for the
identification of molecules which can interact with Bcl-2-like
proteins and inhibit their pro-survival function.
SUMMARY OF THE INVENTION
[0012] Throughout this specification, unless the context requires
otherwise, the word "comprise", and variations such as "comprises"
and "comprising", will be understood to imply the inclusion of a
stated integer or step or group of integers or steps but not the
exclusion of any other integer or step or group of integers or
steps.
[0013] Abbreviations used herein are defined in Table 1.
[0014] The present invention provides small molecule antagonists of
pro-survival molecules and in particular small molecule antagonists
of one or more members of the Bcl-2 family of pro-survival
molecules or other related pro-survival molecules. The generation
and/or selection of the antagonists is based on mimetics of the
natural antagonists of Bcl-2 family proteins (the BH3-only
proteins) and/or mimetics of the structural similarities between
the Bcl-2 molecules which are only inhibited by a narrow range of
BH3-only proteins. Structural studies have revealed that the
hydrophobic face of the amphipathic .alpha.-helix formed by the BH3
domain of the pro-apoptotic proteins inserts into a hydrophobic
groove formed by the BH1, BH2 and BH3 domains of the pro-survival
proteins and inhibits their pro-survival functions. The
.alpha.-helix in the pro-apoptotic proteins comprises hydrophobic
regions on the outer sections of the helical terms referred to as
H1, H2, H3 and H4. The amino acid sequence forms a heptad repeat.
The H1 through H4 outer faces interact with pockets (grooves) on
the Bcl-2 proteins.
[0015] In accordance with the present invention, the BH3-only
proteins are distinguishable functionally with respect to the
spectrum of Bcl-2 target molecules to which they interact. These
groups are classified in accordance with the present invention as
promiscuous and restrictive. By identifying the amino acid charge,
size, conformation, solubility, polarity, hydrophobicity,
hydrophilicity and contribution to tertiary structure differences
between the promiscuous and restrictive BH3-only proteins,
especially surrounding the interaction of the .alpha.-helix of the
BH3 domain and the hydrophobic grooves of the Bcl-2 proteins,
mimetics can be generated or selected which mimic one or the other
of these groups. The Bcl-2 proteins also have structural features
which contribute to the promiscuous or restrictive activity of the
BH3-only protein.
[0016] For example, amino acids in the heptads repeat on the
pro-apoptotic molecule may be modified to reduce an ability for the
amino acid or proximal amino acids in the tertiary structure to fit
into a pocket formed by the tertiary structure on the Bcl-2
proteins.
[0017] Restrictive BH3-only proteins provide, therefore, a scaffold
having a conformation conferring a selective ability to antagonize
particular Bcl-2 proteins. The scaffold may be used in accordance
with the present invention as a template to design mimetics or to
model compounds including promiscuous BH3-only proteins to generate
antagonists with a restrictive binding spectrum.
[0018] Accordingly, in one embodiment of the present invention,
mimetics are made to the restrictive BH3-only proteins enabling
apoptosis to be induced in selected types of cells such as, but not
limited to, cancer cells.
[0019] In yet another embodiment of the present invention, the
promiscuous and restrictive BH3-only proteins differ in relation to
the level of interaction with binding grooves present on the
pro-survival Bcl-2 proteins.
[0020] The present invention also provides computational methods
for predicting the conformation of a molecule which mimics a
restrictive BH3-only protein scaffold to generate and/or select
and/or screen candidate agents which may then be made and evaluated
experimentally for their capacity to induce apoptosis.
[0021] The present invention provides in another embodiment, a
method for generating or selecting an antagonist of the
pro-survival Bcl-2 protein family said method comprising selecting
a scaffold BH3-only protein structure with residue positions
defining an amphipathic .alpha.-helix formed by the BH3 domain;
selecting one or more residue positions associated with a
promiscuous binding phenotype of a BH3-only protein; substituting
amino acid residues conferring a promiscuous phenotype for amino
acids or their chemical analogs which confer a restrictive binding
pattern to a Bcl-2 protein; and analyzing the interaction of each
substitution for an ability to induce a more restrictive spectrum
of binding to a Bcl-2 protein.
[0022] The present invention further provides a method for
generating or selecting an antagonist of the pro-survival Bcl-2
protein family said method comprising selecting a restrictive
BH3-only protein as a scaffold protein, determining the
conformation of the scaffold conferring the restrictive phenotype
and generating or screening for a chemical compound which mimics
said scaffold and/or conformational part conferring a restrictive
spectrum of binding to a Bcl-2 protein.
[0023] The present invention provides, therefore, a computational
method for designing an antagonist of the pro-survival Bcl-2
protein family based on a scaffold BH3-only protein with residue
positions conferring a restrictive phenotype the method comprising
selecting a collection of promiscuous BH3-only proteins; providing
a sequence alignment of these proteins and comparing same to a
restrictive BH3-only protein; generating a frequency of occurrence
for individual amino acids in one or a plurality of positions with
said alignments conferring promiscuity or restrictivity with
respect to binding to Bcl-2 proteins; creating a scoring fimction
selected from charge, size, conformation, solubility, polarity,
hydrophobicity, hydrophilicity and contribution to tertiary
structure using said frequencies; using said scoring function
and/or at least one additional scoring function to generate a set
of optimized protein sequences or their conformational equivalents
and generating or selecting a compound or protein having a
restrictive binding phenotype to a Bcl-2 protein.
[0024] In yet another embodiment, the present invention provides
the use of a promiscuous BH3-only protein in the generation or
selection of amino acid substitution variants which confer a
restrictive binding phenotype to said BH3-only protein or its
chemical or conformational equivalent.
[0025] Still a further aspect of the present invention contemplates
a method for generating or selecting an antagonist of a Bcl-2
protein, said method comprising determining a series of parameters
selected from: [0026] (1) identifying structural dissimilarities
between promiscuous and restrictive Bcl-2 proteins; [0027] (2)
identifying structural dissimilarities between promiscuous and
restrictive BH3-only proteins; and [0028] (3) identifying
structural features of Bcl-2-BH3-only protein complexes and then
designing a mimetic of a BH3-only protein which binds to a
restrictive range of Bcl-2 proteins.
[0029] Mimetics of the BH3-only proteins may also be generated or
selected by methods such as, but not limited to, in silico
screening, high throughput chemical screening, function-based
assays or structure-activity relationships.
[0030] In still yet another embodiment, the BH3-only mimetics of
the present invention are conveniently provided in medicament form
such as a pharmaceutical composition.
[0031] The mimetics of the present invention are particularly
useful in treating subjects with cancer or a propensity to develop
cancer.
BRIEF DESCRIPTION OF THE FIGURES
[0032] FIG. 1 is a graphical representation showing competitive
binding assays. (A) Mcl-1 was injected onto sensor chips with
mutant .sup.4EBimBH3 (blue) or .sup.wtBimBH3 (red) immobilized. To
obtain absolute binding (black), the baseline response with
.sup.4EBimBH3 was subtracted from that with .sup.wtBimBH3. (B)
Pro-survival proteins bind Bim equally. Sensorgrams showing
comparable responses when pro-survival proteins were injected onto
immobilized .sup.wtBimBH3. (C) Solution competition assay.
Increasing competitor peptide concentration (2) decreases
BCl-x.sub.L binding to the immobilized ligand (1). (D)
Pre-incubation with a competitor BH3 peptide diminishes biosensor
responses. Bcl-x.sub.L was pre-incubated with increasing
concentrations of BikBH3 before the mixture was injected over
.sup.wtBimBH3 chip. The line (at 430 s) indicates the response used
for calculating the IC50. (E) BikBH3 effectively competed with
immobilized BimBH3 for Bcl-x.sub.L binding. The relative response
(%) indicates the proportion of Bcl-x.sub.L that still binds the
immobilized peptide in the presence of indicated concentrations of
BikBH3, compared to Bcl-x.sub.L without BikBH3 (100%). (Irregular
responses after 500 s are due to washing the chips after analyte
dissociation). Color reproductions are available from the patentees
on request.
[0033] FIG. 2 is a graphical representation showing pro-apoptotic
BH3-only and pro-survival Bcl-2-like proteins have distinctive
interactions. (A) Using competitive binding assays, the IC50 (nM)
for the indicated interactions were determined. The results shown
are from representative experiments; the variation observed in
multiple experiments was less than two-fold (using different chips
or protein batches). (B) Relative binding affinities of
interactions (tabulated in A) were inversely plotted. The BH3
binding profiles of pro-survival proteins are shown. In (C), the
sequences of human Bcl-2 (residues 93-202), human Bcl-x.sub.L
(86-195), human Bcl-w (42-151), mouse Mcl-1 (190-300) and mouse A1
(33-148) that form their BH3 binding grooves (.alpha.-helices 2 to
8; Hinds et al., EMBO J 22:1497-1507, 2003) were compared and
presented as a phylogenetic tree. Color reproductions are available
from the patentees on request.
[0034] FIG. 3 is a photographical representation showing Bad, Bik
and Noxa have selective pro-survival targets in mammalian cells.
Interactions between FLAG (FL)-tagged pro-survival proteins (human
Bcl-2, human Bcl-x.sub.L and mouse Mcl-1) and HA-tagged BH3-only
proteins (A, human Bim; B, human Puma; C, human Bik; D and E, mouse
Bad; F and G, mouse Noxa) were tested by co-immunoprecipitation.
Equivalent .sup.35S-labeled lysates harvested from 293T cells were
immunoprecipitated with antibodies to the HA, FLAG (FL) or control
(C) tags. Bim (A) or Puma (B) bound Bcl-2, Bcl-x.sub.L and Mcl-1
well. In the top panel of A, Bim.sub.L was used instead of
Bim.sub.EL to discriminate Bim from Bcl-2 by size. (C) Bik
preferentially bound Bcl-x.sub.L. (D) Bad bound Bcl-2 and
Bcl-x.sub.L but not Mcl-1, as confirmed by immunoblotting the same
filters with the indicated antibodies (E). **Endogenous 14-3-3,
associating with Bad, as confirmed by immunoblotting (E). (F) Noxa
only bound Mcl-1, as confirmed by immunoblotting (G). *Degradation
product of Mcl-1 incapable of binding.
[0035] FIG. 4 is a graphical representation showing differential
targeting of pro-survival Bcl-2-like proteins by the BH3-only
proteins. The affinities of BH3-only peptides for pro-survival
proteins (tabulated in FIG. 2) were inversely plotted to allow
comparison of the BH3 domains. Color reproductions are available
from the patentees on request.
[0036] FIG. 5 is a graphical representation showing BH3 peptides
have a propensity to be .alpha.-helical. (A) CD spectra of BH3
peptides and horse heart myoglobin in 30 mM sodium phosphate (pH
7), showing that the BH3 peptides used were largely unstructured
(some have a low % of helicity) whereas the control protein horse
heart myoglobin formed an .alpha.-helical structure as expected
under the buffer condition. Minima (arrowed) at 208 nM and 222 nM
are typical of polypeptides that are .alpha.-helical. (B) CD
spectra of BH3 peptides and myoglobin in 20 mM sodium phosphate (pH
7) supplemented with 30% (v/v) TFE, showing that all BH3 peptides
have an .alpha.-helical conformation similar to that of horse heart
myoglobin in the presence of the helix stabilizing solvent TFE
(Nelson and Kallenbach, Biochemistiy 28:5256-5261, 1989). Color
reproductions are available from the patentees on request.
[0037] FIG. 6 is a graphical representation showing pro-apoptotic
Bad and Noxa target selective pro-survival Bcl-2-like proteins. The
biosensor response to (A) BadBH3 or (B) NoxaBH3-immobilized chips
when recombinant pro-survival Bcl-2-like proteins (Bcl-2
.DELTA.C22, Bcl-x.sub.L .DELTA.C24, Bcl-w .DELTA.C29, Mcl-1
.DELTA.N151 .DELTA.C23, A1 .DELTA.C20) or irrelevant proteins (GST
and LIF-receptor) was injected. Mcl-1 and A1 had no affinity for
BadBH3 whereas Bcl-2, Bcl-x.sub.L, Bcl-w bind BadBH3 avidly (A). A
complementary pattern was observed with NoxaBH3 (B). Color
reproductions are available from the patentees on request.
[0038] FIG. 7 is a graphical representation showing that BH3-only
proteins that bind selective targets have weak killing activity.
(A) The affinities of BH3-only peptides for pro-survival proteins
(tabulated in FIG. 3A) were inversely plotted to facilitate
comparison of the patterns of BH3 binding. (B) Potent killing of
MEFs by Bim and Puma, but not Bmf, Bad, Bik, Hrk or Noxa.
Immortalized 3T9 MEFs were infected with retroviruses expressing
only GFP (control), or one of the BH3-only proteins and GFP. The
viability of the infected (GFP.sup.+ve) cells was determined by PI
exclusion 24 hours after infection. The histograms represent
means.+-.1SD of at least 3 experiments. (C) Interactions between
FLAG (FL)-tagged pro-survival proteins (Bcl-x.sub.L and Mcl-1) and
Bims (or its variants) were tested by co-immunoprecipitation.
Equivalent lysates from transfected 293T cells were
immunoprecipitated with antibodies to Bim, FLAG (FL) tag or a
control irrelevant antigen (C). The filter was probed with a rat
monoclonal anti-FLAG antibody. *Degradation product of Mcl-1. (D)
Bim.sub.s variants that have restricted binding to the pro-survival
proteins are weak killers. Viability of MEFs infected with
retroviruses encoding the indicated proteins (GFP.sup.+ve) was
analyzed 24 hours after infection. The histograms represent
means.+-.1SD of at least 3 experiments. (E) Long-term survival of
MEFs infected with BH3 expressing retroviruses. One hundred
retrovirally-infected cells were plated and the absolute number of
GFP.sup.+ve colonies formed after 6 days scored. No colonies were
obtained after infection with Bims (.dagger.), whereas Bim.sub.s 4E
did not affect long-term viability. Bik, Noxa, Bim.sub.s BadBH3 or
Bim.sub.s NoxaBH3 had modest effects. The data represent the
average number of GFP.sup.+ve colonies formed.+-.1SD from at least
3 experiments.
[0039] FIG. 8 is a graphical representation showing cooperation
between different classes of BH3-only proteins. (A) Based on the
binding data, a model proposed to explain the weak killing activity
of certain BH3-only proteins that have selective targets. (B)
Cooperation between pro-apoptotic BH3-only proteins. MEFs were
infected with retroviruses co-expressing a BH3-only protein
(Bim.sub.s, Bik, Noxa or Noxa3E) and GFP, or a BH3-only protein and
GFP-tagged-Bim.sub.s, -Bim.sub.s BadBH3 or -Bim.sub.s NoxaBH3. Cell
viability was scored 24 hours after infection; data represent
means.+-.1SD of at least 3 experiments.
[0040] FIG. 9 is a representation showing that a less selective
Noxa mutant is a potent killer. (A) Interactions between the
.alpha.-helical BimBH3 region; the numbering refers to mouse
Bim.sub.L) with the target groove of Bcl-x.sub.L (key residues
labeled in black). (B) Alignment of the core BH3 regions of human
Bim (Bim.sub.L) and human Noxa. Mutated Noxa residues are boxed.
(C) Increased binding of Noxa mutants to Bcl-x.sub.L and Bcl-w.
Wild-type or mutant Noxa peptides were tested in solution
competition assays for their capacity to bind Bcl-2, Bcl-x.sub.L,
Bcl-w or Mcl-1. The histograms show the IC50 (nM) for each
interaction. .dagger.: IC50 >100 .mu.M. (D) Noxa m3 binds both
Bcl-x.sub.L and Mcl-1. Interactions between Bcl-x.sub.L or Mcl-1,
and Bim.sub.s NoxaBH3 m3 were tested by co-immunoprecipitation.
*Mcl-1 degradation product. (E) NoxaBH3 m3 is a potent killer.
Survival of MEFs 24 hours after infection with the indicated
retroviruses. (F) Dose-dependent killing by NoxaBH3 m3. Viability
of MEFs infected with NoxaBH3 or NoxaBH3 m3 sorted for low, medium
or high GFP expression; data in (E, F) represent means.+-.1SD of at
least 3 experiments.
DETAILED DESCRIPTION OF THE INVENTION
[0041] The present invention contemplates methods for generating
mimetics of BH3-only proteins proposed to be useful in inducing
apoptosis of selected cells and in particular cancer cells. It is
proposed that amino acid charge, size, conformation, solubility,
polarity, hydrophobicity, hydrophilicity and contribution to
tertiary structure similarities between restrictive BH3-only
proteins and their respective target Bcl-2 proteins be exploited to
generate mimetics of the BH3-only proteins which induce
apoptosis.
[0042] Before describing the subject invention in detail it is to
be noted that the instant invention is not limited to specific
therapeutic components, manufacturing methods, dosage regimens, or
the like, as such may vary. It is also to be understood that the
terminology used herein is for the purpose of describing particular
embodiments only and is not intended to be limiting.
[0043] It must also be noted that, as used in the subject
specification, the singular forms "a", "an" and "the" include
plural aspects unless the context clearly dictates otherwise. Thus,
for example, reference to a "therapeutic agent" includes a single
agent, as well as two or more therapeutic agents; reference to a
"method" includes a single method, as well as two or more methods;
a "residue" includes a single residue, as well as two or more
residues, and so forth.
[0044] Reference herein to "apoptosis" means a form of cell death
in which a programmed sequence of events leads to the elimination
of cells.
[0045] Accordingly, in one embodiment of the present invention,
mimetics are made to the restrictive BH3-only proteins enabling
apoptosis to be induced in selected types of cells such as, but not
limited to, cancer cells.
[0046] Reference herein to "cancer cell" means any cell that
exhibits abnormal growth and which tends to proliferate in an
uncontrolled way and, in some cases, to metastasize. Cancers
contemplated herein include, but are not limited to, ABL1
protooncogene, AIDS Related Cancers, Acoustic Neuroma, Acute
Lymphocytic Leukaemia, Acute Myeloid Leukaemia, Adenocystic
carcinoma, Adrenocortical Cancer, Agnogenic myeloid metaplasia,
Alopecia, Alveolar soft-part sarcoma, Anal cancer, Angiosarcoma,
Aplastic Anaemia, Astrocytoma, Ataxia-telangiectasia, Basal Cell
Carcinoma (Skin), Bladder Cancer, Bone Cancers, Bowel cancer, Brain
Stem Glioma, Brain and CNS Tumors, Breast Cancer, CNS Tumors,
Carcinoid Tumors, Cervical Cancer, Childhood Brain Tumors,
Childhood Cancer, Childhood Leukaemia, Childhood Soft Tissue
Sarcoma, Chondrosarcoma, Choriocarcinoma, Chronic Lymphocytic
Leukaemia, Chronic Myeloid Leukaemia, Colorectal Cancers, Cutaneous
T-Cell Lymphoma, Dermatofibrosarcoma-protuberans,
Desmoplastic-Small-Round-Cell-Tumor, Ductal Carcinoma, Endocrine
Cancers, Endometrial Cancer, Ependymoma, Esophageal Cancer, Ewing's
Sarcoma, Extra-Hepatic Bile Duct Cancer, Eye Cancer, Eye: Melanoma,
Retinoblastoma, Fallopian Tube cancer, Fanconi Anaemia,
Fibrosarcoma, Gall Bladder Cancer, Gastric Cancer, Gastrointestinal
Cancers, Gastrointestinal-Carcinoid-Tumor, Genitourinary Cancers,
Germ Cell Tumors, Gestational-Trophoblastic-Disease, Glioma,
Gynaecological Cancers, Haematological Malignancies, Hairy Cell
Leukaemia, Head and Neck Cancer, Hepatocellular Cancer, Hereditary
Breast Cancer, Histiocytosis, Hodgkin's Disease, Human
Papillomavirus, Hydatidiform mole, Hypercalcemia, Hypopharynx
Cancer, IntraOcular Melanoma, Islet cell cancer, Kaposi's sarcoma,
Kidney Cancer, Langerhan's-Cell-Histiocytosis, Laryngeal Cancer,
Leiomyosarcoma, Leukaemia, Li-Fraumeni Syndrome, Lip Cancer,
Liposarcoma, Liver Cancer, Lung Cancer, Lymphedema, Lymphoma,
Hodgkin's Lymphoma, Non-Hodgkin's Lymphoma, Male Breast Cancer,
Malignant-Rhabdoid-Tumor-of-Kidney, Medulloblastoma, Melanoma,
Merkel Cell Cancer, Mesothelioma, Metastatic Cancer, Mouth Cancer,
Multiple Endocrine Neoplasia, Mycosis Fungoides, Myelodysplastic
Syndromes, Myeloma, Myeloproliferative Disorders, Nasal Cancer,
Nasopharyngeal Cancer, Nephroblastoma, Neuroblastoma,
Neurofibromatosis, Nijmegen Breakage Syndrome, Non-Melanoma Skin
Cancer, Non-Small-Cell-Lung-Cancer-(NSCLC), Ocular Cancers,
Oesophageal Cancer, Oral cavity Cancer, Oropharynx Cancer,
Osteosarcoma, Ostomy Ovarian Cancer, Pancreas Cancer, Paranasal
Cancer, Parathyroid Cancer, Parotid Gland Cancer, Penile Cancer,
Peripheral-Neuroectodermal-Tumors, Pituitary Cancer, Polycythemia
vera, Prostate Cancer, Rare-cancers-and-associated-disorders, Renal
Cell Carcinoma, Retinoblastoma, Rhabdomyosarcoma, Rothmund-Thomson
Syndrome, Salivary Gland Cancer, Sarcoma, Schwannoma, Sezary
syndrome, Skin Cancer, Small Cell Lung Cancer (SCLC), Small
Intestine Cancer, Soft Tissue Sarcoma, Spinal Cord Tumors,
Squamous-Cell-Carcinoma-(skin), Stomach Cancer, Synovial sarcoma,
Testicular Cancer, Thymus Cancer, Thyroid Cancer,
Transitional-Cell-Cancer-(bladder),
Transitional-Cell-Cancer-(renal-pelvis-/-ureter), Trophoblastic
Cancer, Urethral Cancer, Urinary System Cancer, Uroplakins, Uterine
sarcoma, Uterus Cancer, Vaginal Cancer, Vulva Cancer,
Waldenstrom's-Macroglobulinemia, Wilms' Tumor.
[0047] Cancers that are particular targets of the present invention
are those which produce an excess amount of a Bcl-2 protein or
pro-survival relative and/or a reduced amount of a pro-apoptotic
molecule which inhibits a Bcl-2 protein.
[0048] In yet another embodiment of the present invention, the
BH3-only proteins may be promiscuous or restrictive. Reference
herein to "promiscuous" means the protein binds to a number of
targets (i.e. binds to all or multiple Bcl-2 proteins). Reference
herein to "restrictive" means the protein binds only to specific
targets (i.e. binds to only one or a few Bcl-2 proteins). The
promiscuous and restrictive BH3-only proteins may differ in
relation to the level of interaction with binding grooves present
on the pro-survival Bcl-2 proteins.
[0049] In accordance with the present invention, the term "target"
is used to identify a Bcl-2 protein such as Bcl-2, Bcl-x.sub.L,
Bcl-w, Mcl and A1 or any other pro-survival molecule comprising
three or four Bcl-2 homology (BH) regions.
[0050] A "target binder" is used to describe a molecule and or
mimetic BH3-only proteins and which inhibit the pro-survival
proteins. Naturally occurring target binders include Bim, Puma,
Bmf, Bad, Bik, Hrk, Bid and Noxa.
[0051] The aim of the present invention is to generate or select
highly restrictive and specific mimetics which will act as target
binders to inhibitors of apoptosis of particular cells such as
cancer cells.
[0052] The present invention provides in another embodiment, a
method for generating or selecting an antagonist of the
pro-survival Bcl-2 protein family said method comprising selecting
a scaffold BH3-only protein structure with residue positions
defining an amphipathic .alpha.-helix formed by the BH3 domain;
selecting one or more residue positions associated with a
promiscuous binding phenotype of a BH3-only protein; substituting
amino acid residues for each of the residues conferring a
promiscuous phenotype for an amino acid or its chemical analog
which confers a restricted binding phenotype to a Bcl-2 protein;
and analyzing the interaction of each substitution for an ability
to induce a more restrictive spectrum of binding to a Bcl-2
protein.
[0053] Reference herein to a "scaffold protein" means a protein
(i.e. BH3 -only protein) for which a library of variants is
desired. The scaffold protein is used as input in the protein
design calculations, and often is used to facilitate experimental
library generation. A scaffold protein may be any protein that has
a known structure or for which a structure may be calculated,
estimated, modeled or determined experimentally.
[0054] The present invention further provides a method for
generating or selecting an antagonist of the pro-survival Bcl-2
protein family said method comprising selecting a restrictive
BH3-only protein as a scaffold protein, determining the
conformation of the scaffold conferring the restrictive phenotype
and generating or screening for a chemical compound which mimics
said scaffold and/or conformational part conferring a restrictive
spectrum of binding to a Bcl-2 protein.
[0055] In yet another embodiment, the present invention provides
the use of a promiscuous BH3-only protein as an amino acid residue
substitute matrix in the generation or selection of substitute
variants conferring a restrictive binding phenotype to said
BH3-only protein or its chemical or conformational equivalent.
[0056] In one example, the molecular basis for selectivity of the
Noxa BH3-only protein is described.
[0057] Noxa BH3 selectively binds Mcl-1 and A1 and does not bind
Bcl-2, Bcl-w or Bcl-x.sub.L.
[0058] A study of the sequences of the BH3 domain of Noxa reveals
that the amino acid immediately before H4 is uniquely a basic amino
acid in human Noxa and in the two murine Noxa BH3 domains (Table
3). It is proposed that restoration of an acidic residue at that
position, as is more commonly found in other BH3 domain sequences,
restores binding to Bcl-2, Bcl-w and Bcl-x.sub.L. Assay of a mutant
human Noxa BH3 domain in which the relevant lysine amino acid is
replaced by glutamic acid shows an IC50 for the mutant peptide of
5.8 .mu.M, i.e. at least 17 fold tighter than the wild-type
peptide.
[0059] A further unique property of the human Noxa BH3 domain is
the presence of an aromatic amino acid, phenylalanine at the H3
position. This is the only occurrence of an amino acid with a
branched gamma carbon atom (Table 3) and suggests a requirement for
more space in the target Bcl-2 family protein to receive the larger
amino acid at this position. When the amino acid sequences of the
Bcl-2 family proteins are placed in alignment, it is evident that
Mcl-1 and A1 contain smaller amino acids in the receptor site for
the H3 amino acid of the BH3 domain. This conclusion is possible by
drawing on the published three-dimensional structure of Bcl-x.sub.L
complexed with the Bim BH3 domain (Liu, X. et al., Immunity
19:341-352, 2003) and using the sequence alignment referred to
above. Mutation of human Noxa BH3 from F to I at the H3 position
results in an IC50 of 1.1 .mu.M for the mutant peptide, i.e. at
least 90 fold tighter than wild type. The double mutant, K to E
plus F to I shows the changes to be synergistic with an IC50 of 0.1
.mu.M.
[0060] This illustrates how the subject invention enables the
conversion of a selective BH3 domain into a promiscuous BH3
domain.
[0061] Reference herein to an "agent" should be understood as a
reference to any proteinaceous or nonproteinaceous molecule derived
from natural, recombinant or synthetic sources. Useful sources
include the screening of naturally produced libraries, chemical
molecule libraries as well as combinatorial libraries, phage
display libraries and in vitro translation-based libraries.
Particularly useful sources are the modification of a promiscuous
BH3 only protein scaffold to generate a restrictive molecule.
[0062] In one embodiment, the agents of the present invention
useful for the complete suppression of, or substantial decrease in,
the levels or activity of the pro-survival functions of Bcl-2 or a
pro-survival relative may be proteinaceous or chemical molecules.
All such decreases, inhibitions, reductions and down-regulations of
a Bcl-2 family protein pro-survival activity are encompassed by the
terms "antagonist" or "antagonism" or "antagonizing".
[0063] In relation to agents which are proteinaceous molecules,
such molecules include peptides, polypeptide and proteins. In
addition, the terms mutant, part, derivative, homolog, analog or
mimetic are meant to encompass various forms of an agent which
completely suppresses or substantially decreases the pro-survival
functions of Bcl-2 family protein.
[0064] The agents may be naturally occurring or artificially
generated molecules. The agents may be BH-3 only proteins
comprising one or more amino acid substitutions, deletions or
additions. Agents may be generated by mutagenesis or other chemical
methods or generated recombinantly or synthetically. Alanine
scanning is a useful technique for identifying important amino
acids (Wells, Methods Enzymol 202:2699-2705, 1991). In this
technique, an amino acid residue is replaced by Alanine and its
effect on the peptide's activity is determined. Each of the amino
acid residues of the agent is analyzed in this manner to determine
the important structural and/or charge and/or conformational and/or
hydrophobic/hydrophilic regions. Agents are tested for their
ability to bind to Bcl-2 and for other qualities such as longevity,
binding affinity, dissociation rate, ability to cross membranes or
ability to induce apoptosis.
[0065] Agents of the present invention may also encompass Bcl-2
binding portions of a full-length BH3-only protein. Portions are at
least 1, at least 10, least 20 and at least 30 contiguous amino
acids, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30 amino
acids which define a Bcl-2 binding fragment such as an amphipathic
.alpha.-helix structure. It is proposed that this structure
interacts with the hydrophobic grooves of the Bcl-2 proteins.
Peptides of this type may be obtained through the application of
standard recombinant nucleic acid techniques or synthesized using
conventional liquid or solid phase synthesis techniques. For
example, reference may be made to solution synthesis or solid phase
synthesis as described, for example, in Chapter 9 entitled "Peptide
Synthesis" by Atherton and Shephard which is included in a
publication entitled "Synthetic Vaccines" edited by Nicholson and
published by Blackwell Scientific Publications. Alternatively,
peptides can be produced by digestion of an amino acid sequence of
the invention with proteinases such as endoLys-C, endoArg-C,
endoGlu-C and staphylococcus V8-protease. The digested fragments
can be purified by, for example, high performance liquid
chromatographic (HPLC) techniques. Any such fragment, irrespective
of its means of generation, is to be understood as being
encompassed by the term "antagonist" as used herein. Thus
antagonists may comprise a derivative of a promiscuous BH3-only
protein. Such a derivative includes parts, mutants, homologs,
fragments, analogues as well as hybrid or fusion molecules and
glycosylation variants of a promiscuous BH3-only protein.
Derivatives also include molecules having a percent amino acid
sequence identity over a window of comparison after optimal
alignment. Preferably, the percentage similarity between a
particular sequence and a reference sequence is at least about 60%
or at least about 70% or at least about 80% or at least about 90%
or at least about 95% or above such as at least about 96%, 97%,
98%, 99% or greater. Preferably, the percentage similarity between
species, functional or structural homologs of the instant agents is
at least about 60% or at least about 70% or at least about 80% or
at least about 90% or at least about 95% or above such as at least
about 96%, 97%, 98%, 99% or greater. Percentage similarities or
identities between 60% and 100% are also contemplated such as 60,
61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,
78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,
95, 96, 97, 98, 99 or 100%.
[0066] Analogs of residues in a protein antagonist such as a
derivative of a BH3-only protein contemplated herein include but
are not limited to modification to side chains, incorporating
unnatural amino acids and/or their derivatives during peptide,
polypeptide or protein synthesis and the use of crosslinkers and
other methods which impose conformational constraints on the
proteinaceous molecule or their analogs. This term also does not
exclude modifications of the polypeptide, for example,
glycosylations, acetylations, phosphorylations and the like.
Included within the definition are, for example, polypeptides
containing one or more analogs of an amino acid (including, for
example, unnatural amino acids such as those given in Table 1) or
polypeptides with substituted linkages. Such polypeptides may need
to be able to enter the cell.
[0067] Examples of side chain modifications contemplated by the
present invention include modifications of amino groups such as by
reductive alkylation by reaction with an aldehyde followed by
reduction with NaBH.sub.4; amidination with methylacetimidate;
acylation with acetic anhydride; carbamoylation of amino groups
with cyanate; trinitrobenzylation of amino groups with 2, 4,
6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups
with succinic anhydride and tetrahydrophthalic anhydride; and
pyridoxylation of lysine with pyridoxal-5-phosphate followed by
reduction with NaBH.sub.4.
[0068] The guanidine group of arginine residues may be modified by
the formation of heterocyclic condensation products with reagents
such as 2,3-butanedione, phenylglyoxal and glyoxal.
[0069] The carboxyl group may be modified by carbodiimide
activation via 0-acylisourea formation followed by subsequent
derivitisation, for example, to a corresponding amide.
[0070] Sulphydryl groups may be modified by methods such as
carboxymethylation with iodoacetic acid or iodoacetamide; performic
acid oxidation to cysteic acid; formation of a mixed disulphides
with other thiol compounds; reaction with maleimide, maleic
anhydride or other substituted maleimide; formation of mercurial
derivatives using 4-chloromercuribenzoate,
4-chloromercuriphenylsulphonic acid, phenylmercury chloride,
2-chloromercuri-4-nitrophenol and other mercurials; carbamoylation
with cyanate at alkaline pH.
[0071] Tryptophan residues may be modified by, for example,
oxidation with N-bromosuccinimide or alkylation of the indole ring
with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides. Tyrosine
residues on the other hand, may be altered by nitration with
tetranitromethane to form a 3-nitrotyrosine derivative.
[0072] Modification of the imidazole ring of a histidine residue
may be accomplished by alkylation with iodoacetic acid derivatives
or N-carbethoxylation with diethylpyrocarbonate.
[0073] Examples of incorporating unnatural amino acids and
derivatives during peptide synthesis include, but are not limited
to, use of norleucine, 4-amino butyric acid, 4-amino-3-hydroxy
-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine,
norvaline, phenylglycine, ornithine, sarcosine,
4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or
D-isomers of amino acids. A list of unnatural amino acids,
contemplated herein is shown in Table 1. Such unnatural amino acids
may be useful in conferring a tertiary structure analogous to a
restrictive BH3 only protein scaffold.
TABLE-US-00001 TABLE 1 CODES FOR NON-CONVENTIONAL AMINO ACIDS
Non-conventional Non-conventional amino acid Code amino acid Code
.alpha.-aminobutyric acid Abu L-N-methylalanine Nmala
.alpha.-amino-.alpha.-methylbutyrate Mgabu L-N-methylarginine Nmarg
aminocyclopropane- Cpro L-N-methylasparagine Nmasn carboxylate
L-N-methylaspartic acid Nmasp aminoisobutyric acid Aib
L-N-methylcysteine Nmcys aminonorbornyl- Norb L-N-methylglutamine
Nmgln carboxylate L-N-methylglutamic acid Nmglu cyclohexylalanine
Chexa L-Nmethylhistidine Nmhis cyclopentylalanine Cpen
L-N-methylisolleucine Nmile D-alanine Dal L-N-methylleucine Nmleu
D-arginine Darg L-N-methyllysine Nmlys D-aspartic acid Dasp
L-N-methylmethionine Nmmet D-cysteine Dcys L-N-methylnorleucine
Nmnle D-glutamine Dgln L-N-methylnorvaline Nmnva D-glutamic acid
Dglu L-N-methylornithine Nmorn D-histidine Dhis
L-N-methylphenylalanine Nmphe D-isoleucine Dile L-N-methylproline
Nmpro D-leucine Dleu L-N-methylserine Nmser D-lysine Dlys
L-N-methylthreonine Nmthr D-methionine Dmet L-N-methyltryptophan
Nmtrp D-ornithine Dorn L-N-methyltyrosine Nmtyr D-phenylalanine
Dphe L-N-methylvaline Nmval D-proline Dpro L-N-methylethylglycine
Nmetg D-serine Dser L-N-methyl-t-butylglycine Nmtbug D-threonine
Dthr L-norleucine Nle D-tryptophan Dtrp L-norvaline Nva D-tyrosine
Dtyr .alpha.-methyl-aminoisobutyrate Maib D-valine Dval
.alpha.-methyl-.gamma.-aminobutyrate Mgabu D-.alpha.-methylalanine
Dmala .alpha.-methylcyclohexylalanine Mchexa
D-.alpha.-methylarginine Dmarg .alpha.-methylcylcopentylalanine
Mcpen D-.alpha.-methylasparagine Dmasn
.alpha.-methyl-.alpha.-napthylalanine Manap
D-.alpha.-methylaspartate Dmasp .alpha.-methylpenicillamine Mpen
D-.alpha.-methylcysteine Dmcys N-(4-aminobutyl)glycine Nglu
D-.alpha.-methylglutamine Dmgln N-(2-aminoethyl)glycine Naeg
D-.alpha.-methylhistidine Dmhis N-(3-aminopropyl)glycine Norn
D-.alpha.-methylisoleucine Dmile N-amino-.alpha.-methylbutyrate
Nmaabu D-.alpha.-methylleucine Dmleu .alpha.-napthylalanine Anap
D-.alpha.-methyllysine Dmlys N-benzylglycine Nphe
D-.alpha.-methylmethionine Dmmet N-(2-carbamylethyl)glycine Ngln
D-.alpha.-methylornithine Dmorn N-(carbamylmethyl)glycine Nasn
D-.alpha.-methylphenylalanine Dmphe N-(2-carboxyethyl)glycine Nglu
D-.alpha.-methylproline Dmpro N-(carboxymethyl)glycine Nasp
D-.alpha.-methylserine Dmser N-cyclobutylglycine Ncbut
D-.alpha.-methylthreonine Dmthr N-cycloheptylglycine Nchep
D-.alpha.-methyltryptophan Dmtrp N-cyclohexylglycine Nchex
D-.alpha.-methyltyrosine Dmty N-cyclodecylglycine Ncdec
D-.alpha.-methylvaline Dmval N-cylcododecylglycine Ncdod
D-N-methylalanine Dnmala N-cyclooctylglycine Ncoct
D-N-methylarginine Dnmarg N-cyclopropylglycine Ncpro
D-N-methylasparagine Dnmasn N-cycloundecylglycine Ncund
D-N-methylaspartate Dnmasp N-(2,2-diphenylethyl)glycine Nbhm
D-N-methylcysteine Dnmcys N-(3,3-diphenylpropyl)glycine Nbhe
D-N-methylglutamine Dnmgln N-(3-guanidinopropyl)glycine Narg
D-N-methylglutamate Dnmglu N-(1-hydroxyethyl)glycine Nthr
D-N-methylhistidine Dnmhis N-(hydroxyethyl))glycine Nser
D-N-methylisoleucine Dnmile N-(imidazolylethyl))glycine Nhis
D-N-methylleucine Dnmleu N-(3-indolylyethyl)glycine Nhtrp
D-N-methyllysine Dnmlys N-methyl-.gamma.-aminobutyrate Nmgabu
N-methylcyclohexylalanine Nmchexa D-N-methylmethionine Dnmmet
D-N-methylornithine Dnmorn N-methylcyclopentylalanine Nmcpen
N-methylglycine Nala D-N-methylphenylalanine Dnmphe
N-methylaminoisobutyrate Nmaib D-N-methylproline Dnmpro
N-(1-methylpropyl)glycine Nile D-N-methylserine Dnmser
N-(2-methylpropyl)glycine Nleu D-N-methylthreonine Dnmthr
D-N-methyltryptophan Dnmtrp N-(1-methylethyl)glycine Nval
D-N-methyltyrosine Dnmtyr N-methyla-napthylalanine Nmanap
D-N-methylvaline Dnmval N-methylpenicillamine Nmpen
.gamma.-aminobutyric acid Gabu N-(p-hydroxyphenyl)glycine Nhtyr
L-t-butylglycine Tbug N-(thiomethyl)glycine Ncys L-ethylglycine Etg
penicillamine Pen L-homophenylalanine Hphe L-.alpha.-methylalanine
Mala L-.alpha.-methylarginine Marg L-.alpha.-methylasparagine Masn
L-.alpha.-methylaspartate Masp L-.alpha.-methyl-t-butylglycine
Mtbug L-.alpha.-methylcysteine Mcys L-methylethylglycine Metg
L-.alpha.-methylglutamine Mgln L-.alpha.-methylglutamate Mglu
L-.alpha.-methylhistidine Mhis L-.alpha.-methylhomophenylalanine
Mhphe L-.alpha.-methylisoleucine Mile N-(2-methylthioethyl)glycine
Nmet L-.alpha.-methylleucine Mleu L-.alpha.-methyllysine Mlys
L-.alpha.-methylmethionine Mmet L-.alpha.-methylnorleucine Mnle
L-.alpha.-methylnorvaline Mnva L-.alpha.-methylornithine Morn
L-.alpha.-methylphenylalanine Mphe L-.alpha.-methylproline Mpro
L-.alpha.-methylserine Mser L-.alpha.-methylthreonine Mthr
L-.alpha.-methyltryptophan Mtrp L-.alpha.-methyltyrosine Mtyr
L-.alpha.-methylvaline Mval L-N-methylhomophenylalanine Nmhphe
N-(N-(2,2-diphenylethyl) Nnbhm N-(N-(3,3-diphenylpropyl) Nnbhe
carbamylmethyl)glycine carbamylmethyl)glycine
1-carboxy-1-(2,2-diphenyl- Nmbc ethylamino)cyclopropane
[0074] Crosslinkers can be used, for example, to stabilize 3D
conformations, using homo-bifunctional crosslinkers such as the
bifunctional imido esters having (CH.sub.2).sub.n spacer groups
with n=1 to n=6, glutaraldehyde, N-hydroxysuccinimide esters and
hetero-bifunctional reagents which usually contain an
amino-reactive moiety such as N-hydroxysuccinimide and another
group specific-reactive moiety such as maleimido or dithio moiety
(SH) or carbodiimide (COOH). In addition, peptides can be
conformationally constrained by, for example, incorporation of
C.sub..alpha. and N.sub..alpha.-methylamino acids, introduction of
double bonds between C.sub..alpha. and C.sub..beta. atoms of amino
acids and the formation of cyclic peptides or analogs by
introducing covalent bonds such as forming an amide bond between
the N and C termini, between two side chains or between a side
chain and the N or C terminus.
[0075] Reference to a mimetic of a BH3-only protein includes a
target binder (i.e. a BH3-only protein) at the structural and/or
functional level and inhibits a pro-survival Bcl-2-protein. In
accordance with one embodiment of the present invention, it is
proposed to generate selected BH3-only protein mimetics. A BH3-only
protein mimetic is designed based on structural differences between
targets and structural differences between target binders. The
latter may, in accordance with the present invention and as defined
hereinbefore, be divided into promiscuous (i.e. binds to all or
multiple Bcl-2 proteins) or restrictive (i.e. binds to one or only
a few Bcl-2 proteins).
[0076] A peptide mimetic may be a peptide-containing molecule that
mimics elements of protein secondary structure (Johnson et al.,
Peptide Turn Mimetics in Biotechnology and Pharmacy, Pezzuto et
al., Eds., Chapman and Hall, New York, 1993). The underlying
rationale behind the use of peptide mimetics is that the peptide
backbone of proteins exists chiefly to orient amino acid side
chains in such a way as to facilitate molecular interactions such
as those of antibody and antigen, enzyme and substrate or
scaffolding proteins. A peptide mimetic is designed to permit
molecular interactions similar to the natural molecule. Peptide or
non-peptide mimetics of a BH3-only protein may be useful in the
present invention as an agent which decreases the pro-survival
function of Bcl-2.
[0077] The designing of mimetics to a pharmaceutically active
compound is a known approach to the development of pharmaceuticals
based on a "lead" compound. This might be desirable where the
active compound is difficult or expensive to synthesize or where it
is unsuitable for a particular method of administration, e.g.
peptides are unsuitable active agents for oral compositions as they
tend to be quickly degraded by proteases in the alimentary canal.
Mimetic design, synthesis and testing is generally used to avoid
randomly screening large numbers of molecules for a target
property.
[0078] There are several steps commonly taken in the design of a
mimetic from a compound having a given target property. First, the
particular parts of the compound that are critical and/or important
in determining the target property are determined. In the case of a
peptide, this can be done by systematically varying the amino acid
residues in the peptide, e.g. by substituting each residue in turn.
As described hereinbefore, Alanine scans of peptides are commonly
used to refine such peptide motifs. These parts or residues
constituting the active region of the compound are known as its
"pharmacophore".
[0079] Once the pharmacophore has been found, its structure is
modelled according to its physical properties, e.g.
stereochemistry, bonding, size and/or charge, using data from a
range of sources, e.g. spectroscopic techniques, x-ray diffraction
data and NMR. Computational analysis, similarity mapping (which
models the charge and/or volume of a pharmacophore, rather than the
bonding between atoms) and other techniques can be used in this
modelling process.
[0080] In a variant of this approach, the three-dimensional
structure of the ligand and its binding partner are modelled. This
can be especially useful where the ligand and/or binding partner
change conformation on binding, allowing the model to take account
of this in the design of the mimetic. Modelling can be used to
generate inhibitors which interact with the linear sequence or a
three-dimensional configuration.
[0081] A template molecule is then selected onto which chemical
groups which mimic the pharmacophore can be grafted. The template
molecule and the chemical groups grafted onto it can conveniently
be selected so that the mimetic is easy to synthesize, is likely to
be pharmacologically acceptable, and does not degrade in vivo,
while retaining the biological activity of the lead compound.
Alternatively, where the mimetic is peptide-based, further
stability can be achieved by cyclizing the peptide, increasing its
rigidity. The mimetic or mimetics found by this approach can then
be screened to see whether they have the target property, or to
what extent they exhibit it. Further optimization or modification
can then be carried out to arrive at one or more final mimetics for
in vivo or clinical testing.
[0082] The goal of rational drug design in accordance with the
present invention is to use computational methods to generate
and/or select structural analogs of restrictive BH3-only proteins
in order to fashion drugs which are, for example, more active or
stable forms of the polypeptide and which have a restrictive
binding spectrum. In one approach, one first determines the
three-dimensional structure of a protein of interest by x-ray
crystallography, by computer modelling or most typically, by a
combination of approaches. Useful information regarding the
structure of a polypeptide may also be gained by modelling based on
the structure of homologous proteins. An example of rational drug
design is the development of HIV protease inhibitors (Erickson et
al., Science 249:527-533, 1990).
[0083] One method of drug screening utilizes eukaryotic or
prokaryotic host cells which are stably transformed with
recombinant polynucleotides expressing the polypeptide or fragment,
preferably in competitive binding assays. Such cells, either in
viable or fixed form, can be used for standard binding assays. One
may measure, for example, the formation of complexes between a
target or fragment and the agent being tested, or examine the
degree to which the formation of a complex between a target or
fragment and a known ligand is aided or interfered with by the
agent being tested.
[0084] The screening procedure includes assaying (i) for the
presence of a complex between the drug and the target, or (ii) an
alteration in the expression levels of nucleic acid molecules
encoding the target. One form of assay involves competitive binding
assays. In such competitive binding assays, the target is typically
labeled. Free target is separated from any putative complex and the
amount of free (i.e. uncomplexed) label is a measure of the binding
of the agent being tested to target molecule. One may also measure
the amount of bound, rather than free, target. It is also possible
to label the compound rather than the target and to measure the
amount of compound binding to target in the presence and in the
absence of the drug being tested.
[0085] Another technique for drug screening provides high
throughput screening for compounds having suitable binding affinity
to a target and is described in detail in Geysen (International
Patent Publication No. WO 84/03564). Briefly stated, large numbers
of different small peptide test compounds are synthesized on a
solid substrate, such as plastic pins or some other surface. The
peptide test compounds are reacted with a target and washed. Bound
target molecule is then detected by methods well known in the art.
This method may be adapted for screening for non-peptide, chemical
entities. This aspect, therefore, extends to combinatorial
approaches to screening for target antagonists or agonists.
[0086] Purified target can be coated directly onto plates for use
in the aforementioned drug screening techniques. However,
non-neutralizing antibodies to the target may also be used to
immobilize the target on the solid phase. The target may
alternatively be expressed as a fusion protein with a tag
conveniently chosen to facilitate binding and identification.
[0087] In another embodiment, high throughput chemical screening
(HTCS) for inhibitors of Bcl-2 and Bcl-w can be carried out. Given
the interaction of a BH3-only protein like Bim with a pro-survival
molecule (Bcl-2 or Bcl-w) precipitates apoptosis, libraries can be
screened for small organic molecules that bind to the pro-survival
proteins in such a way as to prevent BH3 binding. Multiple
screening campaigns can be undertaken in order to identify
compounds that target one or both anti-apoptotic molecules.
[0088] The proteins necessary for the high capacity assays may be
produced in bacteria and initial studies using an optical biosensor
(BiaCore) show that a biotinylated Bim BH3 peptide binds
His.sub.6-tagged Bcl-w .DELTA.C10 with high affinity
(K.sub.d.about.11 nM) (Hinds et al., EMBO J 22:1497-1507, 2003).
The high capacity binding assays necessary for HTCS have been
developed using AlphaScreen.TM. (Amplified Luminescent Proximity
Homogeneous Assay) technology (Glickman et al., J Biomol Screen
7:3-10, 2002). By revealing changes in fluorescence output as two
partner proteins interact, it can monitor protein interactions with
exquisite sensitivity. AlphaScreen.TM. is well suited for HTCS, as
it is robust and can readily be carried out in small volumes as a
homogenous assay with great dynamic range.
[0089] In one embodiment His.sub.6 Bcl-w .DELTA.C10 is bound to
nickel-coated acceptor beads and the biotinylated BimBH3 peptide is
bound to the streptavidin-coated donor beads. The beads are then
incubated with the test compounds in the wells of a 384-well
microtitre plate (one test compound per well) and the assay results
read using the Fusion alpha plate reader. The binding assay may be
optimized with respect to the concentration of the protein partners
and beads, incubation times and assay volumes so that the assay
typically yields a signal to background ratio of >30:1. The
assay has been validated as the IC.sub.50 values obtained for a
series of peptides were comparable with those obtained using an
optical biosensor. Although the affinities of the peptides spanned
over 3-orders of magnitude (8 nM-35 .mu.M), the strong correlation
observed between the two sets of results (R.sup.2=0.9983) indicates
that the assays measure the same interactions. The binding assays
for His.sub.6 Bcl-2 .DELTA.C22/Bim BH3 may also be optimized. Once
the assay is optimised, it could be subjected to a rigorous quality
control to assess plate-to-plate and day-to-day reproducibility.
Each assay could then be used to screen a unique discovery library.
To eliminate false positives, all inhibitory compounds that meet
the target potency (IC50 <25 .mu.M) may be validated in
secondary competition assays (AlphaScreen.TM., fluorescence
polarisation and BiaCore optical biosensor). The optical biosensor
facilitates to quantify the interactions between Bcl-2 family
members, and ready comparison between the affinities of strong
candidates to the physiological binding by BH3-only proteins can be
made.
[0090] Compounds that pass these initial tests may be checked for
identity and purity by, inter alia, liquid chromatography-mass
spectrometry and then tested for their target specificity, i.e.
affinity for Bcl-2, Bcl-x.sub.L, Bcl-w. Active compounds will also
be tested in assays designed to predict intestinal absorption
(Wohnsland et al., J Med Chem 44:923-930, 2001) and hepatotoxicity.
In addition, in silico methods may be used to predict their
bio-distribution properties, and to exclude pharmacophores that
could present metabolic or toxicity problems (Drug Metabolism
Databases and High-Throughput Testing During Drug Design and
Development, Ed Erhardt, Blackwell Science, Malden, Mass., USA,
1999). The data on all the active compounds may be ranked by
potency in binding assays, target selectivity, favourable
predictive ADMET (Adsorption, Distribution, Metabolism, Excretion
and Toxicity) properties (van de Waterbeemd and Gifford, Nat Rev
Drug Disc 2:192-204, 2003) and chemical tractability. Then, all
available close structural analogues of the top compounds may be
obtained and tested for inhibitory activity in binding and killing
assays to determine preliminary structure-activity relationships
for each structural series.
[0091] In respect of assays on lead compounds for biological
activity, when promising leads are found, their activity on cell
viability in culture may be assessed. Up to 50 lead compounds,
optimised according to the criteria described above, may be tested
on a panel of cultured Tumorigenic and non-Tumorigenic cell lines,
as well as primary mouse and human cell populations, e.g.
lymphocytes. Cell viability may be monitored over 3-7 days of
incubation with 1 nM-100 .mu.M of the compounds. Greatest attention
will, of course, be given to compounds that kill Tumor cells much
more efficiently than their normal cell counterparts. Compounds
that kill at <10 .mu.M may be evaluated for the specificity of
their targets and mode of action. Verifying their mode of action is
important, because a test compound might well kill cells
indirectly. For example, if a lead compound binds with high
selectivity to Bcl-2, it should not kill cells lacking Bcl-2.
Hence, the specificity of action may be confirmed by comparing the
activity of the compound in wild-type cells with those lacking
Bcl-2.
[0092] The most promising candidates may be subjected to a thorough
analysis of their anti-Tumor efficacy in mouse models. In two
models that have fully characterised previously, immuno-competent
mice injected with B-cell lymphomas, derived from either myc
transgenic mice (Adams et al., Nature 318:533-538, 1985) or
myc/bcl-2 doubly transgenic animals (Strasser et al., Supra),
succumb rapidly and reproducibly to a leukaemia/lymphoma syndrome.
Although both tumors respond to standard chemotherapy
(cyclophosphamide), mice injected with myc/bcl-2 Tumor cells
invariably relapse. These two transplantable Tumors will allow
testing of any compounds, given alone or in combination with
cyclophosphamide, in treating these malignancies which closely
model human lymphomas.
[0093] In respect of structure-activity relationships (SAR) of the
lead compounds and their optimisation, the leads selected from
initial screens may require considerable modification to enhance
their biochemical, biological and pharmacological properties
(Bleicher et al., Nat Rev Drug Discov 2:369-378, 2003). To aid
optimisation of these compounds, their mode of action may be
verified in biochemical and structural studies. Furthermore,
complexes formed between the agents and the pro-survival molecules
may be analysed by NMR spectroscopy. Because NMR can detect ligands
of low affinity and reveal where on the target protein they bind,
it can greatly aid the optimisation of binding and accelerate the
drug discovery process (Hajduk et al., J Med Chem 42:2315-2317,
1999; Pellecchia et al., Nat Rev Drug Discov 1:211-219, 2002).
Using techniques such as chemical shift mapping, binding of test
compounds to Bcl-2 proteins will be monitored and those mimicking a
BH3 domain will be selected for optimisation.
[0094] In a related approach, molecular modelling of the lead
agents may be performed to assess their binding in silico using an
adapted DOCK program (Kuntz, Science 257:1078-1082, 1992). Lead
compounds will be modelled onto the target Bcl-2 groove and scoring
functions used to predict the most likely binding modes. This will
guide the design of derivatives that provide additional
interactions to enhance binding. The availability of NMR-derived
experimental data also makes it possible to dock the ligand and the
target flexibly in order to predict improved ligands (Lugovskoy et
al., J Am Chem Soc 124:1234-1240, 2002).
[0095] This information and those from biological assays may be
used to synthesise derivative compounds for further testing. For
each class of lead compound, a strategy for synthesising
derivatives. For example, a typical hit compound is composed of two
or three linked ring systems, each of which may be substituted by a
range of functional groups. By systematically replacing each of the
functional groups, compounds with a wide range of chemical
properties can be made and tested.
[0096] The present invention also provides computational methods
for predicting the conformation of a molecule which mimics a
restrictive BH3-only protein scaffold to generate and/or select
and/or screen candidate agents which may then be made and evaluated
experimentally for their capacity to induce apoptosis.
[0097] The present invention provides, therefore, a computational
method for designing an antagonist of the pro-survival Bcl-2
protein family based on a scaffold BH3-only protein with residue
positions conferring a restrictive phenotype the method comprising
selecting a collection of promiscuous BH3-only proteins; providing
a sequence alignment of these proteins and comparing same to a
restrictive BH3-only protein; generating a frequency of occurrence
for individual amino acids in one or a plurality of positions with
said alignments conferring promiscuity or restrictivity with
respect to binding to Bcl-2 proteins; creating a scoring function
selected from charge, size, conformation, solubility, polarity,
hydrophobicity, hydrophilicity and contribution to tertiary
structure using said frequencies; using said scoring finction and
at least one additional scoring function to generate a set of
optimized protein sequences or their conformational equivalents and
generating or selecting a compound or protein having a restrictive
binding phenotype to a Bcl-2 protein.
[0098] An assessment of the ability of a restrictive BH3-only
protein to antagonize a Bcl-2 protein and induce apoptosis is
important for selection of an appropriate therapeutic protocol.
Such an assessment is suitably facilitated with the assistance of a
computer programmed with software, which inter alia adds a scoring
function (SF) for at least one feature associated with the
restrictive BH3-only protein to provide a potency value (P.sub.A)
corresponding to the degree of Bcl-2 antagonism induced. The SF can
be selected from, inter alia, (a) the number and position of acidic
residues; or (b) the number and position of basic residues; or (c)
the number and position of polar residues; or (d) the number and
position of non-polar residues; or (e) the number and position of
charged residues; or (f) the number and position of uncharged
residues; or (g) the number and position of hydrophillic residues;
or (h) the number and position of hydrophobic residues; or (i) the
levels of residues; or (j) the solubility levels of residues; or
(k) the size of residues; or (l) the contribution to tertiary
structure the residue makes in the BH3-only protein. Thus, in
accordance with the present invention, SF for such features are
stored in a machine-readable storage medium, which is capable of
processing the data to provide a P.sub.A for a particular
restrictive BH3-only protein or chemical equivalent.
[0099] Thus, in another aspect, the invention contemplates a
computer program product for determining the structure of an agent
to induce apoptosis in a cell, said product comprising: [0100] (1)
code that receives as input scoring finction (SF) for at least two
features associated with said BH3-only or Bcl-2 proteins, wherein
said features are selected from, inter alia,: [0101] (a) the number
and position of acidic residues; [0102] (b) the number and position
of basic residues; [0103] (c) the number and position of polar
residues; [0104] (d) the number and position of non-polar residues;
[0105] (e) the number and position of charged residues; [0106] (f)
the number and position of uncharged residues; [0107] (g) the
number and position of hydrophillic residues; [0108] (h) the number
and position of hydrophobic residues; [0109] (i) the levels of
residues; [0110] (j) the solubility levels of residues; [0111] (k)
the size of residues; [0112] (l) the contribution to tertiary
structure the residue makes in the BH3-only protein [0113] (2) code
that adds said SF to provide a sum corresponding to a P.sub.V for
BH3 -only proteins; and [0114] (3) a computer readable medium that
stores the codes.
[0115] In a related aspect, the invention extends to a computer for
assessing the likely usefulness of a BH3-only protein or chemical
equivalent to induce apoptosis in a cell, wherein said computer
comprises: [0116] (1) a machine-readable data storage medium
comprising a data storage material encoded with machine-readable
data, wherein said machine-readable data comprise I.sub.Vs for at
least two features associated with said BH3-only or Bcl-2 proteins,
wherein said features are selected from, inter alia,: [0117] (a)
the number and position of acidic residues; [0118] (b) the number
and position of basic residues; [0119] (c) the number and position
of polar residues; [0120] (d) the number and position of non-polar
residues; [0121] (e) the number and position of charged residues;
[0122] (f) the number and position of uncharged residues; [0123]
(g) the number and position of hydrophillic residues; [0124] (h)
the number and position of hydrophobic residues; [0125] (i) the
levels of residues; [0126] (j) the solubility levels of residues;
[0127] (k) the size of residues; [0128] (l) the contribution to
tertiary structure the residue makes in the BH3-only protein [0129]
(2) a working memory for storing instructions for processing said
machine-readable data; [0130] (3) a central-processing unit coupled
to said working memory and to said machine-readable data storage
medium, for processing said machine readable data to provide a sum
of said SF corresponding to a P.sub.V for said compound(s); and
[0131] (4) an output hardware coupled to said central processing
unit, for receiving said P.sub.V.
[0132] Any general or special purpose computer system is
contemplated by the present invention and includes a processor in
electrical communication with both a memory and at least one
input/output device, such as a terminal. Such a system may include,
but is not limited, to personal computers, workstations or
mainframes. The processor may be a general purpose processor or
microprocessor or a specialized processor executing programs
located in RAM memory. The programs may be placed in RAM from a
storage device, such as a disk or pre-programmed ROM memory. The
RAM memory in one embodiment is used both for data storage and
program execution. The computer system also embraces systems where
the processor and memory reside in different physical entities but
which are in electrical communication by means of a network.
[0133] Agents identified in accordance with the present invention
are useful in the treatment of cancer.
[0134] Reference herein to "treatment" may mean a reduction in the
severity of an existing condition. The term "treatment" is also
taken to encompass "prophylactic treatment" to prevent the onset of
a condition. The term "treatment" does not necessarily imply that a
subject is treated until total recovery. Similarly, "prophylactic
treatment" does not necessarily mean that the subject will not
eventually contract a condition.
[0135] Subject as used herein refers to humans and non-human
primates (e.g. gorilla, macaque, marmoset), livestock animals (e.g.
sheep, cow, horse, donkey, pig), companion animals (e.g. dog, cat),
laboratory test animals (e.g. mouse, rabbit, rat, guinea pig,
hamster), captive wild animals (e.g. fox, deer), reptiles or
amphibians (e.g. cane toad), fish (e.g. zebrafish) and any other
organisms (e.g. c. elegans) who can benefit from the agents of the
present invention. There is no limitation on the type of animal
that could benefit from the presently described agents. The most
preferred subject of the present invention is a human. A subject
regardless of whether it is a human or non-human organism may be
referred to as a patient, individual, animal, host or
recipient.
[0136] Accordingly, another aspect of the present invention
provides a method of preventing or reducing cancer in a subject
said method comprising administering to said subject an effective
amount of an antagonist of a Bcl-2 protein for a time and under
conditions sufficient to prevent or decrease cancer.
[0137] The identification of agents, capable of antagonizing Bcl-2
and inducing apoptosis provides pharmaceutical compositions for use
in the therapeutic treatment of cancer.
[0138] The agents of the present invention can be combined with one
or more pharmaceutically acceptable carriers and/or diluents to
form a pharmacological composition. Pharmaceutically acceptable
carriers can contain a physiologically acceptable compound that
acts to, e.g., stabilize, or increase or decrease the absorption or
clearance rates of the pharmaceutical compositions of the
invention. Physiologically acceptable compounds can include, e.g.,
carbohydrates, such as glucose, sucrose, or dextrans, antioxidants,
such as ascorbic acid or glutathione, chelating agents, low
molecular weight proteins, compositions that reduce the clearance
or hydrolysis of the peptides or polypeptides, or excipients or
other stabilizers and/or buffers. Detergents can also used to
stabilize or to increase or decrease the absorption of the
pharmaceutical composition, including liposomal carriers.
Pharmaceutically acceptable carriers and formulations for peptides
and polypeptide are known to the skilled artisan and are described
in detail in the scientific and patent literature, see e.g.,
Remington's Pharmaceutical Sciences, 18.sup.th Edition, Mack
Publishing Company, Easton, Pa., 1990 ("Remington's").
[0139] Other physiologically acceptable compounds include wetting
agents, emulsifying agents, dispersing agents or preservatives
which are particularly useful for preventing the growth or action
of microorganisms. Various preservatives are well known and
include, e.g., phenol and ascorbic acid. One skilled in the art
would appreciate that the choice of a pharmaceutically acceptable
carrier including a physiologically acceptable compound depends,
for example, on the route of administration of the modulatory agent
of the invention and on its particular physio-chemical
characteristics.
[0140] Administration of the agent, in the form of a pharmaceutical
composition, may be performed by any convenient means known to one
skilled in the art. Routes of administration include, but are not
limited to, respiratorally, intratracheally, nasopharyngeally,
intravenously, intraperitoneally, subcutaneously, intracranially,
intradermally, intramuscularly, intraoccularly, intrathecally,
intracereberally, intranasally, infusion, orally, rectally, patch
and implant.
[0141] For oral administration, the compounds can be formulated
into solid or liquid preparations such as capsules, pills, tablets,
lozenges, powders, suspensions or emulsions. In preparing the
compositions in oral dosage form, any of the usual pharmaceutical
media may be employed, such as, for example, water, glycols, oils,
alcohols, flavoring agents, preservatives, coloring agents,
suspending agents, and the like in the case of oral liquid
preparations (such as, for example, suspensions, elixirs and
solutions); or carriers such as starches, sugars, diluents,
granulating agents, lubricants, binders, disintegrating agents and
the like in the case of oral solid preparations (such as, for
example, powders, capsules and tablets). Because of their ease in
administration, tablets and capsules represent the most
advantageous oral dosage unit form, in which case solid
pharmaceutical carriers are obviously employed. If desired, tablets
may be sugar-coated or enteric-coated by standard techniques. The
active agent can be encapsulated to make it stable to passage
through the gastrointestinal tract while at the same time allowing
for passage across the blood brain barrier, see, e.g, International
Patent Publication Number WO 96/11698.
[0142] Agents of the present invention, when administered orally,
may be protected from digestion. This can be accomplished either by
complexing the nucleic acid, peptide or polypeptide with a
composition to render it resistant to acidic and enzymatic
hydrolysis or by packaging the nucleic acid, peptide or polypeptide
in an appropriately resistant carrier such as a liposome. Means of
protecting compounds from digestion are well known in the art, see,
e.g. Fix, Pharm Res 13:1760-1764, 1996; Samanen et al., J Pharm
Pharmacol 48:119-135, 1996; U.S. Pat. No. 5,391,377, describing
lipid compositions for oral delivery of therapeutic agents
(liposomal delivery is discussed in further detail, infra). The
pharmaceutical forms suitable for injectable use include sterile
aqueous solutions (where water-soluble) or dispersions and sterile
powders for the extemporaneous preparation of sterile injectable
solutions or dispersion or may be in the form of a cream or other
form suitable for topical application. It must be stable under the
conditions of manufacture and storage and must be preserved against
the contaminating action of microorganisms such as bacteria and
fungi. The carrier can be a solvent or dispersion medium
containing, for example, water, ethanol, polyol (for example,
glycerol, propylene glycol and liquid polyethylene glycol, and the
like), suitable mixtures thereof, and vegetable oils. The proper
fluidity can be maintained, for example, by the use of a coating
such as lecithin, by the maintenance of the required particle size
in the case of dispersion and by the use of superfactants. The
prevention of the action of microorganisms can be brought about by
various antibacterial and antifungal agents, for example, parabens,
chlorobutanol, phenol, sorbic acid, thimerosal and the like. In
many cases, it will be preferable to include isotonic agents, for
example, sugars or sodium chloride. Prolonged absorption of the
injectable compositions can be brought about by the use in the
compositions of agents delaying absorption, for example, aluminum
monostearate and gelatin.
[0143] Sterile injectable solutions are prepared by incorporating
the agents in the required amount in the appropriate solvent with
various of the other ingredients enumerated above, as required,
followed by filtered sterilisation. Generally, dispersions are
prepared by incorporating the various sterilised active ingredient
into a sterile vehicle which contains the basic dispersion medium
and the required other ingredients from those enumerated above. In
the case of sterile powders for the preparation of sterile
injectable solutions, the preferred methods of preparation are
vacuum drying and the freeze-drying technique which yield a powder
of the active ingredient plus any additional desired ingredient
from previously sterile-filtered solution thereof.
[0144] For parenteral administration, the agent may dissolved in a
pharmaceutical carrier and administered as either a solution or a
suspension. Illustrative of suitable carriers are water, saline,
dextrose solutions, fructose solutions, ethanol, or oils of animal,
vegetative or synthetic origin. The carrier may also contain other
ingredients, for example, preservatives, suspending agents,
solubilizing agents, buffers and the like. When the agents are
being administered intrathecally, they may also be dissolved in
cerebrospinal fluid.
[0145] For transmucosal or transdermal administration, penetrants
appropriate to the barrier to be permeated can be used for
delivering the agent. Such penetrants are generally known in the
art e.g. for transmucosal administration, bile salts and fusidic
acid derivatives. In addition, detergents can be used to facilitate
permeation. Transmucosal administration can be through nasal sprays
or using suppositories e.g. Sayani and Chien, Crit Rev Ther Drug
Carrier Syst 13:85-184, 1996. For topical, transdermal
administration, the agents are formulated into ointments, creams,
salves, powders and gels. Transdermal delivery systems can also
include patches.
[0146] For inhalation, the agents of the invention can be delivered
using any system known in the art, including dry powder aerosols,
liquids delivery systems, air jet nebulizers, propellant systems,
and the like, see, e.g., Patton, Nat Biotech 16:141-143, 1998;
product and inhalation delivery systems for polypeptide
macromolecules by, e.g., Dura Pharmaceuticals (San Diego, Calif.),
Aradigm (Hayward, Calif.), Aerogen (Santa Clara, Calif.), Inhale
Therapeutic Systems (San Carlos, Calif.), and the like. For
example, the pharmaceutical formulation can be administered in the
form of an aerosol or mist. For aerosol administration, the
formulation can be supplied in finely divided form along with a
surfactant and propellant. In another aspect, the device for
delivering the formulation to respiratory tissue is an inhaler in
which the formulation vaporizes. Other liquid delivery systems
include, for example, air jet nebulizers.
[0147] The agents of the invention can also be administered in
sustained delivery or sustained release mechanisms, which can
deliver the formulation internally. For example, biodegradable
microspheres or capsules or other biodegradable polymer
configurations capable of sustained delivery of a peptide can be
included in the formulations of the invention (e.g. Putney and
Burke, Nat Biotech 16:153-157, 1998).
[0148] In preparing pharmaceuticals of the present invention, a
variety of formulation modifications can be used and manipulated to
alter pharmacokinetics and biodistribution. A number of methods for
altering pharmacokinetics and biodistribution are known to one of
ordinary skill in the art. Examples of such methods include
protection of the compositions of the invention in vesicles
composed of substances such as proteins, lipids (for example,
liposomes, see below), carbohydrates, or synthetic polymers
(discussed above). For a general discussion of pharmacokinetics,
see, e.g., Remington's.
[0149] In one aspect, the pharmaceutical formulations comprising
agents of the present invention are incorporated in lipid
monolayers or bilayers such as liposomes, see, e.g., U.S. Pat. Nos.
6,110,490; 6,096,716; 5,283,185 and 5,279,833. The invention also
provides formulations in which water-soluble modulatory agents of
the invention have been attached to the surface of the monolayer or
bilayer. For example, peptides can be attached to
hydrazide-PEG-(distearoylphosphatidyl) ethanolamine-containing
liposomes (e.g. Zalipsky et al., Bioconjug Chem 6:705-708, 1995).
Liposomes or any form of lipid membrane, such as planar lipid
membranes or the cell membrane of an intact cell e.g. a red blood
cell, can be used. Liposomal formulations can be by any means,
including administration intravenously, transdemially (Vutla et
al., J Pharm Sci 85:5-8, 1996), transmucosally, or orally. The
invention also provides pharmaceutical preparations in which the
nucleic acid, peptides and/or polypeptides of the invention are
incorporated within micelles and/or liposomes (Suntres and Shek, J
Pharm Pharmacol 46:23-28, 1994; Woodle et al., Pharm Res 9:260-265,
1992). Liposomes and liposomal formulations can be prepared
according to standard methods and are also well known in the art
see, e.g., Remington's; Akimaru et al., Cytokines Mol Ther
1:197-210, 1995; Alving et al., Immunol Rev 145:5-31, 1995; Szoka
and Papahadjopoulos, Ann Rev Biophys Bioeng 9:467-508, 1980, U.S.
Pat. Nos. 4, 235,871, 4,501,728 and 4,837,028.
[0150] The pharmaceutical compositions of the invention can be
administered in a variety of unit dosage forms depending upon the
method of administration. Dosages for typical pharmaceutical
compositions are well known to those of skill in the art. Such
dosages are typically advisorial in nature and are adjusted
depending on the particular therapeutic context, patient tolerance,
etc. The amount of agent adequate to accomplish this is defined as
the "effective amount". The dosage schedule and effective amounts
for this use, i.e., the "dosing regimen" will depend upon a variety
of factors, including the stage of the disease or condition, the
severity of the disease or condition, the general state of the
patient's health, the patient's physical status, age,
pharmaceutical formulation and concentration of active agent, and
the like. In calculating the dosage regimen for a patient, the mode
of administration also is taken into consideration. The dosage
regimen must also take into consideration the pharmacokinetics,
i.e., the pharmaceutical composition's rate of absorption,
bioavailability, metabolism, clearance, and the like. See, e.g.,
Remington's; Egleton and Davis, Peptides 18:1431-1439, 1997;
Langer, Science 249:1527-1533, 1990.
[0151] In accordance with these methods, the agents and/or
pharmaceutical compositions defined in accordance with the present
invention may be co-administered with one or more other agents.
Reference herein to "co-administered" means simultaneous
administration in the same formulation or in two different
formulations via the same or different routes or sequential
administration by the same or different routes. Reference herein to
"sequential" administration is meant a time difference of from
seconds, minutes, hours or days between the administration of the
two types of agents and/or pharmaceutical compositions.
Co-administration of the agents and/or pharmaceutical compositions
may occur in any order.
[0152] Alternatively, targeting therapies may be used to deliver
the active agent more specifically to certain types of cell, by the
use of targeting systems such as antibodies or cell specific
ligands or specific nucleic acid molecules. Targeting may be
desirable for a variety of reasons, e.g. if the agent is
unacceptably toxic or if it would otherwise require too high a
dosage or if it would not otherwise be able to enter the target
cells.
[0153] Instead of administering the agents directly, they could be
produced in the target cell, e.g. in a viral vector such as
described above or in a cell based delivery system such as
described in U.S. Pat. No. 5,550,050 and International Patent
Publication Numbers WO 92/19195, WO 94/25503, WO 95/01203, WO
95/05452, WO 96/02286, WO 96/02646, WO 96/40871, WO 96/40959 and WO
97/12635. The vector could be targeted to the target cells. The
cell based delivery system is designed to be implanted in a
patient's body at the desired target site and contains a coding
sequence for the target agent. Alternatively, the agent could be
administered in a precursor form for conversion to the active form
by an activating agent produced in, or targeted to, the cells to be
treated. See, for example, European Patent Application Number 0 425
731A and International Patent Publication Number WO 90/07936.
[0154] In yet another aspect, the present invention provides kits
comprising the compositions e.g. agents of the present invention.
The kits can also contain instructional material teaching the
methodologies and uses of the invention, as described herein.
[0155] Those skilled in the art will appreciate that the invention
described herein is susceptible to variations and modifications
other than those specifically described. It is to be understood
that the invention includes all such variations and modifications.
The invention also includes all of the steps, features,
compositions and compounds referred to or indicated in this
specification, individually or collectively, and any and all
combinations of any two or more of said steps or features.
[0156] The present invention is fuither described by the following
non-limiting examples.
EXAMPLE 1
Experimental Procedures
[0157] The following experimental procedures are used in the
subsequent Examples which follow.
[0158] Expression constructs:
[0159] Human Bcl-2 (Acc. no. NP.sub.--000624; residues 1-217) and
human Bcl-w (Acc. no. NP.sub.--004041; residues 1-164; C29S A128E)
were cloned into pQE-9 (Qiagen); expressed proteins have additional
N-terminal residues (MRGSHHHHHHGS, SEQ ID NO:1). Human Bcl-x.sub.L
(Acc. no. NP.sub.--612815; residues 1-209), mouse Mcl-1 (Acc. no.
NP.sub.--031588; residues 152-308) and mouse A1 (Acc. no.
NP.sub.--033872; residues 1-152) were cloned into pGEX-6P-3
(Amersham Biosciences) such that only five additional
vector-derived residues (GPLGS) were present in the proteins
following PreScission protease digestion (see below). FLAG
(DYKDDDDK, SEQ ID NO:2)-tagged mammalian expression vectors for
human Bcl-2, human Bcl-x.sub.L and mouse Mcl-1 are described in
Huang et al., EMBO J 16:4628-4638, 1997. N-terminally HA
(YPYDVPDYA, SEQ ID NO:3)-tagged full-length human Bim.sub.EL, human
Bim.sub.L, human Puma, mouse Bad, human Bik and mouse Noxa were
sub-cloned into pEF PGKhygro (Huang et al., Supra; O'Conner et al.,
EMBO J 17:384-395, 1998). Proof-reading Pfu polymerase (Stratagene)
was used for PCR and the constructs verified by automated
sequencing. Details of oligonucleotides used and constructs are
available from the inventors.
[0160] The constructs, in pQE-9 (Qiagen) include human (h) Bcl-2
(Acc. no. NP.sub.--000624; residues 1-217), and hBcl-w
(NP.sub.--612815; residues 1-209) with C29S and A128E mutations to
improve its stability (Hinds et al., 2003). The HexaHis tag
(HHHHHH) allowed their purification on a nickel column. Recombinant
hBcl-x.sub.L .DELTA.C24, mouse (m) Mcl-1 .DELTA.N151 .DELTA.C23 and
mA1 .DELTA.C20 were expressed as GST fusion proteins and cleaved
from glutathione-sepharose columns with PreScission protease and
purified as described (Day et al., 1999; Hinds et al., 2003). The
constructs used, in pGEX-6P-3 (Amersham Biosciences), included
sequences from Bcl-x.sub.L (NP.sub.--612815; residues 1-209), Mcl-1
(NP.sub.--031588; residues 152-308), and A1 (NP.sub.--033872;
residues 1-152). After the protease digestion, they retain five
N-terminal vector-derived residues (GPLGS).
[0161] The peptides used in this study (FIG. 2A), which have free
N- and C-termini, were synthesized by Mimotopes (Victoria,
Australia). All peptides were purified by reverse-phase HPLC and
were >90% pure, except for hbik (87%), mBmf (85%), and mNoxa B
(78%). Their identities were confirmed by electrospray mass
spectrometry. The peptides, quantified by weighing and by their
absorbance at 214 nM on an analytical HPLC column, were completely
dissolved as 1-2 mM stock solutions in water; hBimBH3 was dissolved
in DMSO. The accession numbers on which the peptides were based
are: mBim.sub.L (AAC40030), hBim.sub.L (AAC39594), hPuma
(AAK39542), mBmf (AAK38747), hBad (NP.sub.--004313), hBik
(NP.sub.--001188), hHrk (NP.sub.--003797), hBid (NP.sub.--001187),
hNoxa (NP.sub.--066950), mNoxa (NP.sub.--067426).
[0162] Recombinant proteins and peptides:
[0163] Recombinant human Bcl-2 .DELTA.C22 and human Bcl-w
.DELTA.C29 (containing C29S and A128E mutations to improve protein
solubility but not affecting binding characteristics), expressed as
N-terminal HexaHis fusion proteins, were prepared as described in
Wilson-Annan et al., J. Cell Biol 162:877-888, 2003. Recombinant
human Bcl-x.sub.L .DELTA.C24, mouse Mcl-1 .DELTA.N151 .DELTA.C23
and mouse A1 .DELTA.C20 were expressed as GST fusion proteins and
cleaved off Glutathione Sepharose columns as described (Day et al.,
Cell Death Differ 6:1125-1132, 1999; Hinds et al., EMBO J
22:1497-1507, 2003).
[0164] The peptides used in this study, with free N- and C-termini,
were synthesized by Mimotopes. All peptides purified by
reverse-phase HPLC were greater than 90% pure, except for Bik
(87%), m Bmf (85%), and m Noxa2 (78%). Their identities were
confirmed by electrospray Mass Spectrometric analyses. The peptides
were quantified by weighing and by their absorbance at 214 nM on an
analytical HPLC column; they were completely dissolved as 1-2 mM
stock solutions in water. The accession numbers for the peptides
were: mouse Bim.sub.L (AAC40030), human BimL (AAC39594), human Puma
(AAK39542), mouse Bmf (AAK38747), human Bad (NP.sub.--004313),
human Bik (NP.sub.--001188), human Hrk (NP.sub.--003797), human Bid
(NP.sub.--001187), human Noxa (NP.sub.--066950), and mouse Noxa
(NP.sub.--067426).
[0165] Circular dichroism (CD) spectroscopy:
[0166] For the circular dichroism (CD) measurements, the stock
solutions of peptides and horse heart myoglobin were diluted to a
final concentration of 0.15 mg/ml in either 30 mM sodium phosphate
(pH 7) or in 20 mM sodium phosphate (pH 7) supplemented with 30%
(v/v) (2,2,2-Trifluoroethanol) (TFE). CD spectra were recorded at
room temperature on an AVIV 62DS model spectropolarimeter with 0.1
cm cuvette. Two sequential scans were recorded and the background
spectrum of the buffer alone was subtracted.
[0167] Affinity measurements and solution competition assays:
[0168] Affinity measurements were performed at room temperature on
a Biacore 3000 biosensor (Biacore) with HBS (10 mM HEPES pH 7.2,
150 mM NaCl, 3.4 mM EDTA, 0.005% Tween 20) as the running buffer.
Mouse 26-mer .sup.wtBimBH3, .sup.4EBimBH3 mutant, BadBH3, NoxaBH3
or control irrelevant peptides were immobilized onto CM5 sensor
chips using amine-coupling chemistry (Wilson-Annan et al., Supra).
To assess the binding affinities of pro-survival Bcl-2-like
proteins for BimBH3 directly, the proteins were directly injected
into the sensor chip at 20 .mu.l/min. Residual bound proteins were
desorbed with 50 mM NaOH or 6 M GuHCl (pH 7.2), followed by two
washes with running buffer. Binding kinetics were derived from
sensorgrams, following subtraction of baseline responses, using BIA
evaluation software (version 3, Biacore) (Wilson-Annan et al.,
Supra).
[0169] The relative affinities of BH3 peptides for pro-survival
Bcl-2 proteins were assessed by comparing their abilities to
compete with immobilized .sup.wtBimBH3 peptide for binding to
Bcl-2-like proteins (Wilson-Annan et al., Supra). A fixed
sub-saturating amount (10 nM) of a pro-survival Bcl-2 protein was
incubated with varying amounts of competitor BH3 peptide in HBS for
>2 hr on ice. The mixtures were then injected over a sensor chip
containing a channel bearing mouse .sup.wtBimBH3 and a control
channel with mouse .sup.4EBimBH3 immobilized. The baseline response
(control channel) was subtracted to obtain the absolute binding
response. Taking the response for the pro-survival protein alone as
the maximal response (100%), the relative residual binding (%) in
the presence of increasing amounts of competitor peptides at a
given injection time point (430 s) was then calculated. The
relative residual responses (f) were plotted against initial
peptide concentrations and fitted to the equation
f=100/(1+(c/IC.sub.50).sup.m), where c=concentration of competitor
peptide, m=curvature constant, and IC.sub.50=concentration of
competitor peptide required to reduce binding by 50%.
Theoretically, IC50=[A]/2+K.sub.D, where [A] is the analyte
concentration.
[0170] Some of recombinant proteins (Bcl-2, Bcl-x.sub.L, A1)
studied contain cysteine residues but the behavior of Bcl-2 or
Bcl-x.sub.L was not affected by dithiothreitol (DTT). 2.5 mM
Tris-(carboxyethyl)phosphine hydrochloride (TCEP) was included in
the incubation mixtures with A1, which contains two cysteines and
appears to be less stable than the other proteins studied.
[0171] Transient transfection, immunoprecipatation and
immunoblotting:
[0172] The maintenance, transfection and metabolic labeling of the
human embryonic kidney (HEK) 293T cells with
.sup.35S-methionine/cysteine (NEN) as well as
co-immunoprecipitation have been described (Huang et al., Supra;
Moriishi et al., Proc Natl Acad Sci USA 96:9683-9688, 1999;
O'Conner et al., Supra). Briefly, equivalent TCA-precipitable
lysates were immunoprecipiated using the mouse monoclonal
antibodies to HA (HA.11; CRP), FLAG (M2; Sigma) and control Glu-Glu
(CRP) tags. The proteins were resolved by SDS:PAGE, transferred
onto nitrocellulose membranes and detected by fluorography
(Amplify; Amersham Biosciences). Immunoblotting was performed using
rat monoclonal antibodies to HA (3F10; Roche), FLAG (9H1;
(Wilson-Annan et al., Supra) or mouse monoclonal anti-14-3-3.beta.
(H-8; Santa Cruz) detected by HRP-conjugated anti-rat (Southern
Biotechnology) or anti-mouse (Silenus) antibodies. The proteins
were revealed by enhanced chemiluminescence (ECL; Amersham
Biosciences).
EXAMPLE 2
BimBH3 binds Bcl-2, Bcl-x.sub.L, Bcl-w, Mcl-1 and A1 tightly
[0173] Generating the soluble, monomeric and equivalent recombinant
pro-survival proteins needed for comparative in vitro binding
studies required removal of their hydrophobic C-terminal domains
(e.g. Hinds et al., Supra) and the N-terminal PEST region of Mcl-1
(Kozopas et al., Proc Natl Acad Sci 90:3516-3520, 1993). Likewise,
full-length BH3-only proteins could not be reliably produced, so we
used long peptides (24-26 residues; Table 3), because the reduced
helical propensity of shorter ones can reduce binding (Petros et
al., 2000, Supra). Since a 26-mer peptide spanning the BH3 domain
of mouse Bim (BimBH3) binds to Bcl-w as avidly as longer Bim
polypeptides (Wilson-Annan et al., Supra), we used it to measure
the affinity of Bim for the other mammalian pro-survival molecules.
Mcl-1, for example, gave a strong response when run over the
immobilized wild-type but not over a mutant (non-binding) BimBH3
peptide (FIG. 1A). Indeed, Bcl-2, Bcl-x.sub.L, Bcl-w, Mcl-1 and A1
all showed strong 1:1 stoichiometric binding to .sup.wtBimBH3 (FIG.
1B), the dissociation equilibrium constants (K.sub.D) ranging from
0.2 to 4.5 nM (Table 2). Thus, Bim targets all five mammalian
pro-survival proteins comparably.
TABLE-US-00002 TABLE 2 COMPARABLE BINDING OF BIM TO PRO-SURVIVAL
PROTEINS Analyte K.sub.D (nM) k.sub.d (10.sup.-3 s.sup.-1) k.sub.a
(10.sup.3 M.sup.-1 s.sup.-1) Bcl-2 .DELTA.C22 4.5 0.14 30
Bcl-x.sub.L .DELTA.C24 0.8 0.44 570 Bcl-w .DELTA.C29 1.6 2.70 1700
Mcl-1 .DELTA.N151 .DELTA.C23 0.2 0.26 1300 A1 .DELTA.C20 0.5 0.14
290
[0174] The binding constants of the pro-survival molecules for
.sup.wtBimBH3, immobilized on sensor chip, were determined using
biosensor experiments as described in Hinds et al., Supra;
Wilson-Annan et al., Supra.
EXAMPLE 3
Certain BH3 domains bind pro-survival targets selectively
[0175] To assess if other BH3 peptides bind pro-survival proteins
similarly, we directly compared their binding affinities in
solution using a competitive binding assay. In such assays, the
quality and absolute quantity of target proteins are less critical
than in direct binding, and solution binding precludes the steric
hindrance encountered with some immobilized peptides. FIG. 1C
illustrates the approach: pre-incubation of Bcl-x.sub.L in solution
with increasing amounts of BikBH3 reduced its binding to
.sup.wtBimBH3 (FIG. 1D). From the attenuation in binding, the IC50
(competitor peptide concentration that halves binding) of BikBH3
can be calculated (FIG. 1E). As the IC50 values reflect relative
binding affinities (see Experimental Procedures), we used this
assay to compare the binding affinities of eight BH3 peptides
(Table 3) to the five pro-survival proteins. Strikingly, the
relative affinities of the BH3 peptides for different pro-survival
proteins varied over 10,000-fold (FIG. 2A and Table 2). Their
binding properties fell into several classes (FIG. 2B and FIG. 4).
Only BimBH3 and PumaBH3 had comparable affinities for all the
pro-survival proteins. The other BH3 peptides were surprisingly
selective for particular subsets of them. BadBH3, for example,
strongly preferred Bcl-2, BCl-x.sub.L and Bcl-w (5.3 to 30 nM) to
A1 (-15 .mu.M) or Mcl-1 (>100 .mu.M) (FIGS. 2A and B), and
BmfBH3 showed similar preferences. In marked contrast, NoxaBH3 was
highly selective for Mcl-1 and A1 (nM range) but did not bind
detectably to the others (>100 .mu.M). Finally, BikBH3, HrkBH3
and BidBH3 preferred Bcl-x.sub.L, Bcl-w and A1 over Bcl-2 or Mcl-1.
Contrary to the prevailing view, the pro-survival proteins also had
unique patterns for BH3 binding: Bcl-x.sub.L behaved like Bcl-w,
with Bcl-2 more distinct, whereas Mcl-1 and A1 formed a separate
group (FIG. 2B).
TABLE-US-00003 TABLE 3 BH3 PEPTIDES H1 H2 H3 m .sup.wtBimBH3
(83-108) D L R P E I R I A Q E L R R I m .sup.4EBimBH3 (83-108) --
-- -- -- -- -- -- E -- -- -- E -- -- E Bim (81-106) D M R P E I W I
A Q E L R R I Puma (130-155) E E Q W A R E I G A Q L R R M m Bmf
(128-151) H R A E V Q I A R K L Q C I Bad (103-128) N L W A A Q R Y
G R E L R R M Bik (51-75) M E G S D A L A L R L A C I Hrk (26-51) R
S S A A Q L T A A R L K A I Bid (81-104) D I I R N I A R H L A Q V
Noxa (18-43) P A E L E V E C A T Q L R R F m Noxa1 (16-41) R A E L
P P E F A A Q L R K I m Noxa2 (68-93) P A D L K D E C A -- Q L R R
I H1 H2 H3 H4 m .sup.wtBimBH3 (83-108) G D E F N E T Y T R R
.sup.wtBimBH3 m .sup.4EBimBH3 (83-108) -- -- -- E -- -- -- -- -- --
-- .sup.4EBimBH3 Bim (81-106) G D E F N A Y Y A R R Bim Puma
(130-155) A D D L N A Q Y E R R Puma m Bmf (128-151) A D Q F H R L
H T Q m Bmf Bad (103-128) S D E F V D S F K K G Bad Bik (51-75) G D
E M D V S L R A P Bik Hrk (26-51) G D E L H Q R T M W R Hrk Bid
(81-104) G D S M D R S I P P G Bid Noxa (18-43) G D K L N F R Q K L
L Noxa m Noxa1 (16-41) G D K V Y C T W S A P m Noxa1 m Noxa2
(68-93) G D K V N L R Q K L L N m Noxa2 H4
[0176] The immobilized peptides (top 2 rows) were derived from
mouse (m) Bim.sub.L. .sup.4EBimBH3 has four hydrophobic residues
(H1-H4) critical for interacting with the pro-survival proteins
(see text) mutated to glutamic acids (E). Competitor peptides were
derived from human proteins except those denoted "m" (mouse). The
sequences were aligned using the GCG "PILEUP" program as described
in Huang and Strasser, Supra
[0177] Since all the BH3 peptides bound avidly to at least two
pro-survival proteins (FIG. 2), it is unlikely that their integrity
or conformation affected our results. Nevertheless, as the binding
of shorter BadBH3 peptides to Bcl-x.sub.L depended on their
helicity (Petros et al., 2000, Supra), we assessed the conformation
of the peptides used by CD (circular dichroism) spectroscopy. In an
aqueous environment, all the peptides appeared to be largely
unstructured (FIG. 5A). However, on addition of 30% TFE
(2,2,2-Trifluoroethanol), a solvent that stabilizes cc-helix
formation (Nelson and Kallenbach, Biochemistry 28:5256-5261, 1989),
they readily became .alpha.-helical (Supplemental FIG. S2B),
indicating their helical potential. We observed no correlation
between their helical propensity in TFE and binding affinities.
[0178] The distinctive and complementary binding patterns observed
with Bad and Noxa were particularly striking (FIG. 2). The results
with Noxa are consistent with those in another solution competition
assay (Letai et al., Supra), but Noxa reportedly bound to Bcl-2 and
Bcl-x.sub.L in a co-immunoprecipitation assay (Oda et al., Science
288:1053-1058, 2000). To address this discrepancy, we also
performed direct binding assays with immobilized BadBH3 or NoxaBH3
peptides. In accord with the solution competition results, only
Bcl-2, Bcl-x.sub.L and Bcl-w bound to BadBH3 (FIG. 6A) and only
Mcl-1 and A1 to NoxaBH3 (FIG. 6B). Unlike human Noxa, mouse Noxa
contains two BH3 domains (Oda et al., Supra), but both mouse Noxa
BH3 peptides (Table 2) bound Mcl-1 (IC50 87 and 109 nM) but not
Bcl-2, Bcl-x.sub.L or Bcl-w. Therefore, Noxa presumably
specifically antagonizes Mcl-1 and A1.
EXAMPLE 4
Bad, Bik and Noxa Have Selective Physiological Targets in Mammalian
Cells
[0179] The selective interactions were confirmed in different
assays. GST pull-down experiments, performed with the recombinant
BH3-only proteins that could be made (Bim, Bmf, Bad, and Bid), gave
results concordant with the solution competition assays. Most
pertinently, GST Mcl-1 bound tightly to Bim, weakly to Bmf but not
to Bad. To verify the in vitro findings, interactions of selected
pairs of the full-length N-terminally tagged proteins were
investigated in mammalian cells by co-immunoprecipitation. HA-Bim,
Puma, Bad, Bik or Noxa were co-expressed in HEK293T cells with
FLAG-Bcl-2, BCl-x.sub.L and Mcl-1, as representatives of the three
classes of pro-survival proteins profiled (FIG. 2B). The cells were
metabolically labeled with .sup.35S-methionine/cysteine to permit
semi-quantitative assessment of any interaction between
co-associating radiolabeled proteins. For every pair tested, the
interactions detected by co-immunoprecipitation (FIG. 3) concurred
with the prior affinity measurements (FIG. 2). In particular, Bad
bound well to Bcl-2 and Bcl-x.sub.L but not appreciably to Mcl-1
(FIGS. 3D, 3E) (Opferman et al., Nature 426:671-b 676, 2003),
whereas Noxa (full-length mouse Noxa bearing two BH3s) interacted
only with Mcl-1 (FIGS. 3F, 3G). Bik bound Bcl-x.sub.L to a greater
extent than Bcl-2 or Mcl-1 (FIG. 3C), whereas Bim and Puma, as
expected, bound all three pro-survival proteins well (FIGS. 3A and
3B).
EXAMPLE 5
Specific Residues Are Important for Determining Selectivity of Noxa
for Mcl-1
[0180] When the sequences of BH3 domains were compared, it appears
that that F32 and K35 may be important for determining the
specificity of Noxa for Mcl-1. Wild-type Noxa did not bind
Bcl-x.sub.L, but mutation to either F32 or K35 enhances Bcl-x.sub.L
binding that is further enhanced in the double mutant (Table 4).
This strongly suggests that F32 and K35 of Noxa are important for
determining selectivity of Noxa for Mcl-1.
TABLE-US-00004 TABLE 4 SPECIFIC RESIDUES ARE IMPORTANT FOR
DETERMINIG SELECTIVITY OF Noxa FOR Mcl-1 IC50 for Bcl-x.sub.L (nM)
Noxa P A E L E V E C A T Q L R R F G D K L N F R Q K L L
>100,000 (18-43) Noxa P A E L E V E C A T Q L R R F G D E L N F
R Q K L L 5,800 (18-43) mt1 Noxa P A E L E V E C A T Q L R R I G D
K L N F R Q K L L 1,100 (18-43) mt2 Noxa P A E L E V E C A T Q L R
R I G D E L N F R Q K L L 110 (18-43) mt3 .uparw. .uparw.
EXAMPLE 6
Bad, Bik and Noxa Have Distinct Targets in Mammalian Cells
[0181] To verify the in vitro findings, selected pairs of the
full-length N-terminally tagged proteins associated in mammalian
cells were investigated by co-immunoprecipitation. As
representatives of the different classes of pro-survival proteins
profiled (FIG. 4), Bcl-2, Bcl-x.sub.L and Mcl-1 were co-expressed
in 293T cells with Bim, Puma, Bik, Bad or Noxa. The cells were
metabolically labeled with .sup.35S-methionine/cysteine to permit
semi-quantitative assessment of any interaction between the labeled
proteins or interaction was gauged by the more sensitive western
blotting.
[0182] Significantly, for every pair tested, the interactions
detected by co-immunoprecipitation were those expected from the
prior affinity measurements (FIG. 4). In particular, Bad bound well
to Bcl-2 and Bcl-x.sub.L but not appreciably to Mcl-1 whereas Noxa
(full-length mouse Noxa bearing two BH3s) interacted only with
Mcl-1. Furthermore, Bik bound Bcl-x.sub.L to a greater extent than
Bcl-2 or Mcl-1, whereas Bim, as expected, bound avidly to all three
pro-survival proteins, as did Puma.
EXAMPLE 7
BH3-only Proteins With Restricted Targets Are Weaker Killers
[0183] To assess the biological relevance of the selective binding,
retroviral delivery was used to compare the ability of different
BH3-only proteins to kill wild-type mouse embryonic fibroblasts
(MEFs). To monitor expression of the introduced gene, a vector
(pMIG) in which its expression is coupled via an internal ribosomal
entry site (IRES) to that of green fluorescent protein (GFP) was
used (Van Parijs et al., Immunity 11:281-288, 1999). The cells were
efficiently infected (>90%) and the introduced BH3-only proteins
were comparably expressed. Viability of the infected (GFP.sup.+ve)
cells was scored 24 hours after infection (FIGS. 7A, 7B) and in
long-term colony assays (FIG. 7E).
[0184] Whereas infection with the parental virus caused minimal
apoptosis, most cells infected with a virus expressing Puma or
various Bim isoforms (Bim.sub.s, Bim.sub.L or Bim.sub.EL) soon died
(FIG. 7B) in a dose-dependent manner and failed to generate
colonies (FIG. 7E). Importantly, the other BH3-only proteins tested
(Bmf, Bad, Bik, Hrk, Noxa), although adequately expressed were much
less potent killers (FIG. 7B), probably because none of these
BH3-only proteins can neutralize all the pro-survival molecules
(Bcl-2, Bcl-x.sub.L, Mcl-1) expressed substantially in MEFs.
[0185] The weaker pro-apoptotic activity of certain BH3 -only
proteins, has usually been attributed to specific negative
regulatory mechanisms, such as the binding of Bad by 14-3-3
proteins. To preclude such effects and allow direct comparison
between different BH3 domains, chimeric molecules in which
Bim.sub.s BH3 was replaced with that of Puma, Bad or Noxa were also
analysed. Bim.sub.s was chosen as the common backbone because
Bim.sub.s, the most potent Bim isoform is not regulated by
interaction with the dynein motor complex. Its pro-apoptotic
activity relies solely upon the BH3 region, because Bim.sub.s 4E,
which has the BH3 mutated, does not bind any of the pro-survival
molecules and lacks pro-apoptotic function. Another advantage of
the chimeric proteins, unlike their native counterparts, is that
all were expressed at comparable levels.
[0186] Notably, the Bim.sub.s chimera with the PumaBH3 retained
Puma's ability to bind all the pro-survival molecules tested and
killed as potently as native Bim or Puma. In contrast, the
Bim.sub.s chimeras with the BadBH3 or the NoxaBH3 behaved like
native Bad or Noxa respectively. Bim.sub.s BadBH3 bound Bcl-x.sub.L
but not Mcl-1, whereas Bim.sub.s NoxaBH3 instead bound Mcl-1.
Accordingly, these two chimeras exhibited weak pro-apoptotic
activities in both short- and long-term assays. Thus, the killing
induced by each BH3-only protein appears to reflect its ability to
bind tightly to all the pro-survival Bcl-2-like molecules present
in the cell.
EXAMPLE 8
Functional Complementation Between Bad and Noxa
[0187] Since the results show that apoptosis requires
neutralization of all the relevant pro-survival proteins, BH3-only
proteins with complementary binding profiles (FIG. 8A) should
cooperate in killing cells. Thus, the neutralization of Mcl-1 by
Noxa should be complemented by co-expression of a BadBH3 to
neutralize Bcl-2 and Bcl-x.sub.L in MEFs (FIG. 8A). Likewise, the
NoxaBH3 should augment killing induced by Bik, which binds to
Mcl-1.about.40-fold less well than to Bcl-x.sub.L. To allow tests
of complementation, pairs of BH3-only proteins were co-expressed in
the pMIG vector by replacing GFP with GFP fusions of Bim.sub.s, or
of the chimeric Bim.sub.s BadBH3 and Bim.sub.s NoxaBH3. Such
fusions behave like their parental BH3-only counterparts, since a
GFP Bim.sub.s fusion was a potent killer (FIG. 8B).
[0188] Importantly, co-expression of Noxa with the BadBH3 resulted
in as much cell death as expression of Bim.sub.s alone (FIG. 8B).
Similarly, the NoxaBH3 potently enhanced killing by Bik. The
observed synergies reflect complementary BH3 function, because
co-expression of a NoxaBH3 did not augment Noxa killing, and no
significant killing (FIG. 8B) was observed when Bim.sub.s BadBH3
fused to GFP was co-expressed with an inert form of Noxa (the
.sup.3EBH3 mutant; see Experimental Procedures), which does not
bind Mcl-1. Thus, both Bad and Bik, which predominantly bind Bcl-2
and Bcl-x.sub.L, can potently cooperate with Noxa, which
selectively binds Mcl-1, to augment the weak killing observed when
any of the three BH3-only proteins is expressed alone in
fibroblasts.
EXAMPLE 9
Potent Killing Induced by a Promiscuous Noxa Mutant
[0189] The molecular basis of the selective binding profile and
weak killing activity of Noxa was then explored. The functional
complementation results (FIG. 8) suggest that a form of Noxa that
binds additional pro-survival proteins would be a potent killer. A
high resolution X-ray crystallographic structure of the
Bcl-x.sub.L: Bim complex guided the search for such a variant (FIG.
9A). The hydrophobic residues of BimBH3 (including leucine L94,
isoleucine I97, phenylalanine F101; based on mouse Bim.sub.L
numbering) bind hydrophobic pockets on the surface of Bcl-x.sub.L.
The pocket for Bim I97 (h3) on Bcl-x.sub.L (and Bcl-2) is partly
formed by phenylalanine (F97) and tyrosine (Y101) residues, which
are larger than the corresponding residues in Mcl-1 (valine,
histidine) or in A1 (valine, valine), suggesting that Mcl-1 and A1
can more readily accommodate the .gamma.-branched phenylalanine
(F32) found in the human NoxaBH3 (FIG. 9B). Furthermore, a mutual
charge pair is formed between the negatively charged glutamate
(E100) of Bim and positively charged arginine (R100) on
Bcl-x.sub.L, but this arginine, conserved in Bcl-2 and Bcl-w, is
replaced in Mcl-1 by neutral asparagine and in A1 by a glutamate.
Hence, the charge change from glutamate (E100) in the BimBH3 to
lysine (K35) present in Noxa (FIG. 9B) may be a factor that impairs
NoxaBH3 binding to Bcl-x.sub.L.
[0190] Based on these considerations, Noxa peptides with the
mutations K35E (m1), F32I (m2) or both together (m3) (FIG. 9B) were
tested for binding to pro-survival proteins in solution competition
assays. Their binding to Bcl-2 remained weak, but binding to
Bcl-x.sub.L and Bcl-w increased markedly (FIG. 9C). For
Bcl-x.sub.L, the charge switch mutation K35E (m1) increased binding
more than 20 fold; the F32I substitution (m2) enhanced binding more
than 100 fold; and the double mutation (m3) further augmented
binding (FIG. 9C). Whereas the IC50 for wt NoxaBH3 was >100,000
nM for both Bcl-x.sub.L and Bcl-w, the m3 mutant exhibited an IC50
of 110 nM for Bcl-x.sub.L and 410 nM for Bcl-w, while Mcl-1 binding
was slightly improved (FIG. 9C). Co-immunoprecipitation experiments
confirmed that Noxa m3 bound Bcl-x.sub.L in cells (FIG. 9D),
whereas wild-type Noxa did not (FIG. 7C). Thus, the change of two
key BH3 residues sufficed to broaden the specificity of Noxa.
[0191] Significantly, when expressed in fibroblasts, Noxa m3 proved
to be a more potent killer than wild-type Noxa (FIGS. 9E, 9F).
Thus, the binding of Noxa to Mcl-1 alone is not sufficient to kill
MEFs. Efficient induction of apoptosis by Noxa requires additional
BH3 interactions with proteins of the Bcl-2/Bcl-x.sub.L/Bcl-w
class, provided either by co-expression of Bad-like proteins (FIG.
8) or by mutations that decrease Noxa selectivity (m3; FIG. 9).
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Sequence CWU 1
1
3112PRTpeptides 1Met Arg Gly Ser His His His His His His Gly Ser1 5
1028PRTpeptides 2Asp Tyr Lys Asp Asp Asp Asp Lys1 539PRTpeptides
3Tyr Pro Tyr Asp Val Pro Asp Tyr Ala1 5
* * * * *